Heterologous polypeptides expressed in filamentous fungi, processes for making same, and vectors for making same

ABSTRACT

Novel vectors are disclosed for expressing and secreting heterologous polypeptides from filamentous fungi. Such vectors are used in novel processes to express and secrete such heterologous polypeptides. The vectors used for transforming a filamentous fungus to express and secrete a heterologous polypeptide include a DNA sequence encoding a heterologous polypeptide and a DNA sequence encoding a signal sequence which is functional in a secretory system in a given filamentous fungus and which is operably linked to the sequence encoding the heterologous polypeptide. Such signal sequences may be the signal sequence normally associated with the heterologous polypeptides or may be derived from other sources. The vector may also contain DNA sequences encoding a promoter sequence which is functionally recognized by the filamentous fungus and which is operably linked to the DNA sequence encoding the signal sequence. Preferably functional polyadenylation sequences are operably linked to the 3′ terminus of the DNA sequence encoding the heterologous polypeptides. Each of the above described vectors are used in novel processes to transform a filamentous fungus wherein the DNA sequences encoding the signal sequence and heterologous polypeptide are expressed. The thus synthesized polypeptide is thereafter secreted from the filamentous fungus.

[0001] This is a continuation in part of U.S. patent application Ser.No. 771,374, filed Aug. 29, 1985.

FIELD OF THE INVENTION

[0002] The present invention is directed to heterologous polypeptidesexpressed and secreted by filamentous fungi and to vectors and processesfor expressing and secreting such polypeptides. More particularly, theinvention discloses transformation vectors and processes using the samefor expressing and secreting biologically active bovine chymosin andheterologous glucoamylase by a filamentous fungus.

BACKGROUND OF THE INVENTION

[0003] The expression of DNA sequences encoding heterologouspolypeptides (i.e., polypeptides not normally expressed and secreted bya host organism) has advanced to a state of considerable sophistication.For example, it has been reported that various DNA sequences encodingpharmacologically desirable polypeptides [e.g., human growth hormone(1), human tissue plasminogen activator (2), various human interferons(6), urokinase (5), Factor VIII (4), and human serum albumin (3)] andindustrially important enzymes [e.g., chymosin (7), alpha amylases (8),and alkaline proteases (9)] have been cloned and expressed in a numberof different expression hosts. Such expression has been achieved bytransforming prokaryotic organisms [e.g., E. coli (10) or B. subtilis(11)) or eukaryotic organisms [e.g., Saccharomyces cerevisiae (7),Kluyveromyces lactis (12) or Chinese Hamster Ovary cells (2)] with DNAsequences encoding the heterologous polypeptide.

[0004] Some polypeptides, when expressed in heterologous hosts, do nothave the same level of biological activity as their naturally producedcounterparts when expressed in various host organisms. For example,bovine chymosin has very low biological activity when expressed by E.coli (13) or S. cerevisiae (7). This reduced biological activity in E.coli is not due to the natural inability of E. coli to glycosylate thepolypeptide since chymosin is not normally glycosylated (14). Suchrelative inactivity, both in E. coli and S. cerevisiae, however, appearsto be primarily due to improper folding of the polypeptide chain asevidenced by the partial post expression activation of such expressedpolypeptides by various procedures. In such procedures, expressedchymosin may be sequentially denatured and renatured in a number of waysto increase biological activity: e.g., treatment with urea (13),exposure to denaturing/renaturing pH (13) and denaturation and cleavageof disulfide bonds followed by renaturation and regeneration of covalentsulfur linkages (15). Such denaturation/renaturation procedures,however, are not highly efficient [e.g., 30% or less recovery ofbiological activity for rennin (13)], and add considerable time andexpense in producing a biologically active polypeptide.

[0005] Other heterologous polypeptides are preferably expressed inhigher eukaryotic hosts (e.g., mammalian cells). Such polypeptides areusually glycopolypeptides which require an expression host which canrecognize and glycosylate certain amino acid sequences in theheterologous polypeptide. Such mammalian tissue culture systems,however, often do not secrete large amounts of heterologous polypeptideswhen compared with microbial systems. Moreover, such systems aretechnically difficult to maintain and consequently are expensive tooperate.

[0006] Transformation and expression in a filamentous fungus involvingcomplementation of aroD mutants of N. crassa lacking biosyntheticdehydroquinase has been reported (16). Since then, transformation basedon complementation of glutamate dehydrogenase deficient N. crassamutants has also been developed (17). In each case the dehydroquinase(ga2) and glutamate dehydrogenase (am) genes used for complementationwere derived from N. crassa and therefore involved homologousexpression. Other examples of homologous expression in filamentous fungiinclude the complementation of the auxotrophic markers trpC, (18) andargB (19) in A. nidulans and the transformation of A. nidulans toacetamide or acrylamide utilization by expression of the A. nidulansgene encoding acetamidase (20).

[0007] Expression of heterologous polypeptides in filamentous fungi hasbeen limited to the transformation and expression of fungal andbacterial polypeptides. For example, A. nidulans, deficient inorotidine-5′-phosphate decarboxylase, has been transformed with aplasmid containing DNA sequences encoding the pyr4 gene derived from N.crassa (21,32). A. niger has also been transformed to utilize acetamideand acrylamide by expression of the gene encoding acetamidase derivedfrom A. nidulans (22).

[0008] Examples of heterologous expression of bacterial polypeptides infilamentous fungi include the expression of a bacterialphosphotransferase in N. crassa (23) Dictyostellium discoideum (24) andCephalosporium acremonium (25).

[0009] In each of these examples of homologous and heterologous fungalexpression, the expressed polypeptides were maintained intracellularlyin the filamentous fungi.

[0010] Accordingly, an object of the invention herein is to provide forthe expression and secretion of heterologous polypeptides by and fromfilamentous fungi including vectors for transforming such fungi andprocesses for expressing and secreting such heterologous polypeptides.

SUMMARY OF THE INVENTION

[0011] The inventor includes novel vectors for expressing and secretingheterologous polypeptides from filamentous fungi. Such vectors are usedin novel processes to express and secrete such heterologouspolypeptides. The vectors used for transforming a filamentous fungus toexpress and secrete a heterologous polypeptide include a DNA sequenceencoding a heterologous polypeptide and a DNA sequence encoding a signalsequence which is functional in a secretory system in a givenfilamentous fungus and which is operably linked to the sequence encodingthe heterologous polypeptide. Such signal sequences may be the signalsequence normally associated with the heterologous polypeptides or maybe derived from other sources.

[0012] The vector may also contain DNA sequences encoding a promotersequence which is functionally recognized by the filamentous fungus andwhich is operably linked to the DNA sequence encoding the signalsequence. Preferably functional polyadenylation sequences are operablylinked to the 3′ terminus of the DNA sequence encoding the heterologouspolypeptides.

[0013] Each of the above described vectors are used in novel processesto transform a filamentous fungus wherein the DNA sequences encoding thesignal sequence and heterologous polypeptide are expressed. The thussynthesized polypeptide is thereafter secreted from the filamentousfungus.

BRIEF DESCRIPTION OF THE DRAWINGS

[0014]FIG. 1 is a restriction map of the Aspergillus niger glucoamylaseinserts in pGa1 and pGa5.

[0015]FIG. 2 depicts the construction of pDJB-gam-1.

[0016]FIG. 3 depicts the construction of mp19GAPR.

[0017]FIGS. 4, 5, 6, and 7 depict the construction of pGRG1, pGRG2,pGRG3, and pGRG4.

[0018]FIG. 8 shows the strategy used to generate mp19 GAPR^(Δ)C1-^(Δ)C4from mp19 GAPR.

[0019]FIG. 9 depicts the construction of pCR160.

[0020]FIG. 10 is a partial restriction map of the Mucor miehei carboxylprotease gene including 5′ and 3′ flanking sequences.

[0021]FIGS. 11 A,B. and C is the DNA sequence of Mucor miehei, carboxylprotease including the entire coding sequence and 5′ and 3′ flankingsequences.

[0022]FIG. 12 depicts the construction of pMeJB1-7.

[0023]FIGS. 13 A and B are a partial nucleotide and restriction map ofANS-1.

[0024]FIG. 14 depicts the construction of pDJB-3.

[0025]FIG. 15 depicts the construction of plasmid pCJ:GRG1 throughpCJ:GRG4.

[0026]FIG. 16 depicts a restriction endonuclease cleavage map of the3.7Kb BamHI fragment from pRSH1.

[0027]FIG. 17 depicts the construction of pCJ:RSH1 and pCJ:RSH2.

[0028]FIG. 18 depicts the expression of H. grisea glucoamylase from A.nidulans.

DETAILED DESCRIPTION

[0029] The inventors have demonstrated that heterologous polypeptidesfrom widely divergent sources can be expressed and secreted byfilamentous fungi. Specifically, bovine chymosin, glucoamylase fromAspergillus niger and Humicola grises and the carboxyl protease fromMucor miehei have been expressed in and secreted from A. nidulans. Inaddition, bovine chymosin has been expressed and secreted from A.awamori and Trichoderma reesei. Biologically active chymosin wasdetected in the culture medium without further treatment. This resultwas surprising in that the vectors used to transform A. nidulans wereconstructed to secrete prochymosin which requires exposure to an acidicenvironment (approximately pH 2) to produce biologically activechymosin.

[0030] In general, a vector containing DNA sequences encoding functionalpromoter and terminator sequences (including polyadenylation sequences)are operably linked to DNA sequences encoding various signal sequencesand heterologous polypeptides. The thus constructed vectors are used totransform a filamentous fungus. Viable transformants may thereafter beidentified by screening for the expression and secretion of theheterologous polypeptide.

[0031] Alternatively, an expressible selection characteristic may beused to isolate transformants by incorporating DNA sequences encodingthe selection characteristic into the transformation vector. Examples ofsuch selection characteristics include resistance to variousantibiotics, (e.g., aminoglycosides, benomyl etc.) and sequencesencoding genes which complement an auxotrophic defect (e.g. pyr4complementation of pyr4 deficient A. nidulans, A. awamori or Trichodermareesei or ArgB complementation of ArgB deficient A. nidulans or A.awamori) or sequences encoding genes which confer a nutritional (e.g.,acetamidase) or morphological marker in the expression host.

[0032] In the preferred embodiments disclosed a DNA sequence encodingthe ANS-1 sequence derived from A. nidulans is included in theconstruction of the transformation vectors of the present invention.This sequence increases the transformation efficiency of the vector.Such sequences, however, are not considered to be absolutely necessaryto practice the invention.

[0033] In addition, certain DNA sequences derived from the bacterialplasmid pBR325 form part of the disclosed transformation vectors. Thesesequences also are not believed to be necessary for transformingfilamentous fungi. These sequences instead provide for bacterialreplication of the vectors during vector construction. Other plasmidsequences which may also be used during vector construction includepBR322 (ATCC 37017), RK-2 (ATCC 37125), pMB9 (ATCC 37019) and pSC101(ATCC 37032).

[0034] The disclosed preferred embodiments are presented by way ofexample and are not intended to limit the scope of the invention.

[0035] Definitions

[0036] By “expressing polypeptides” is meant the expression of DNAsequences encoding the polypeptide.

[0037] “Polypeptides” are polymers of α-amino acids which are covalentlylinked through peptide bonds. Polypeptides include low molecular weightpolymers as well as high molecular weight polymers more commonlyreferred to as proteins. In addition, a polypeptide can be aphosphopolypeptide, glycopolypeptide or metallopoly-peptide. Further,one or more polymer chains may be combined to form a polypeptide.

[0038] As used herein a “heterologous polypeptide” is a polypeptidewhich is not normally expressed and secreted by the filamentous fungusused to express that particular polypeptide. Heterologous polypeptidesinclude polypeptides derived from prokaryotic sources (e.g., α-amylasefrom Bacillus species, alkaline protease from Bacillus species, andvarious hydrolytic enzymes from Pseudomonas, etc.), polypeptides derivedfrom eukaryotic sources (e.g., bovine chymosin, human tissue plasminogenactivator, human growth hormone, human interferon, urokinase, humanserum albumin, factor VIII etc.), and polypeptides, derived from fungalsources other than the expression host (e.g., glucoamylase from A. nigerand Humicola grisea expressed in A. nidulans, the carboxyl protease fromMucor miehei expressed in A. nidulans, etc.).

[0039] Heterologous polypeptides also include hybrid polypeptides whichcomprise a combination of partial or complete polypeptide sequencesderived from at least two different polypeptides each of which may behomologous or heterologous with regard to the fungal expression host.Examples of such hybrid polypeptides include: 1) DNA sequences encodingprochymosin fused to DNA sequences encoding the A. niger glucoamylasesignal and pro sequence alone or in conjunction with various amounts ofamino-terminal mature glucoamylase codons, and 2) DNA sequences encodingfungal glucoamylase or any fungal carboxy protease, human tissueplasminogen activator or human growth hormone fused to DNA sequencesencoding a functional signal sequence alone or in conjunction withvarious amounts of amino-terminal propeptide condons or mature codonsassociated with the functional signal.

[0040] Further, the heterologous polypeptides of the present inventionalso include: 1) naturally occuring allellic variations that may existor occur in the sequence of polypeptides derived from the aboveprokaryotic, eukaryotic and fungal sources as well as those used to formthe above hybrid polypeptides,, and 2) engineered variations in theabove heterologous polypeptides brought about, for example, by way ofsite specific mutagenesis wherein various deletions, insertions orsubstitutions of one or more of the amino acids in the heterologouspolypeptides are produced.

[0041] A “biochemically active heterologous polypeptide” is aheterologous polypeptide which is secreted in active form as evidencedby its ability to mediate: 1) the biochemical activity mediated by itsnaturally occurring counterpart, or 2) in the case of hybridpolypeptides, the biochemical activity mediated by at least one of thenaturally occurring counterparts comprising the hybrid polypeptides.

[0042] Each of the above defined heterologous polypeptides is encoded bya heterologous DNA sequence which contains a stop signal which isrecognized by the filamentous fungus in which expression and secretionoccurs. When recognized by the host, the stop signal terminatestranslation of the mRNA encoding the heterologous polypeptide.

[0043] The “filamentous fungi” of the present invention are eukaryoticmicroorganisms and include all filamentous forms of the subdivisionEumycotina (26). These fungi are characterized by a vegatative myceliumcomposed of chitin, cellulose, and other complex polysaccharides. Thefilamentous fungi of the present invention are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligately aerobic. In contrast, vegetative growth by yeasts such as S.cerevisiae is by budding of a unicellular thallus, and carbon catabolismmay be fermentative. S cerevisiae has a prominent, very stable diploidphase whereas, diploids exist only briefly prior to meiosis infilamentous fungi like Aspergilli and Neurospora. S. cervisiae has 17chromosomes as opposed to 8 and 7 for A. nidulans and N. crassarespectively. Recent illustrations of differences between S. cerevisiaeand filamentous fungi include the inability of S. cerevisiae to processAspergillus and Trichoderma introns and the inability to recognize manytranscriptional regulators of filamentous fungi (27).

[0044] Various species of filamentous fungi may be used as expressionhosts including the following genera: Aspergillus, Trichoderma,Neurospora, Podospora, Endothia Mucor, Cochiobolus and Pyricularia.Specific expression hosts include A. nidulans (18, 19, 20, 21, 61), A.niger (22), A. awomari, e.g., NRRL 3112, ATCC 22342 (NRRL 3112), ATCC44733, ATCC 14331 and strain UVK 143f, A. oryzae, e.g., ATCC 11490, N.crassa (16, 17, 23), Trichoderma reesei, e.g. NRRL 15709, ATCC 13631,56764, 56765, 56466, 56767, and Trichoderma viride, e.g., ATCC 32098 and32086.

[0045] As used herein, a “promotor sequence” is a DNA sequence which isrecognized by the particular filamentous fungus for expression purposes.It is operably linked to a DNA sequence encoding the above definedpolypeptides. Such linkage comprises positioning of the promoter withrespect to the initiation codon of the DNA sequence encoding the signalsequence of the disclosed transformation vectors. The promoter sequencecontains transcription and translation control sequences which mediatethe expression of the signal sequence and heterologous polypeptide.Examples include the promoter from A. niger glucoamylase (39,48), theMucor miehei carboxyl protease herein, and A. niger α-glucosidase (28),Trichoderma reesei cellobiohydrolase I (29), A. nidulans trpC (18) andhigher eukaryotic promoters such as the SV40 early promoter (24).

[0046] Likewise a “terminator sequence” is a DNA sequence which isrecognized by the expression host to terminate transcription. It isoperably linked to the 3′ end of the DNA encoding the heterologouspolypeptide to be expressed. Examples include the terminator from A.nidulans trpC (18), A. niger glucoamylase (39,48), A. nigerα-glucosidase (28), and the Mucor miehei carboxyl protease herein,although any fungal terminator is likely to be functional in the presentinvention.

[0047] A “polyadenylation sequence” is a DNA sequence which whentranscribed is recognized by the expression host to add polyadenosineresidues to transcribed mRNA. It is operably linked to the 3′ end of theDNA encoding the heterologous polypeptide to be expressed. Examplesinclude polyadenylation sequences from A. nidulans trpC (18), A. nigerglucoamylase (39,48), A. niger α-glucosidase (28), and the Mucor mieheicarboxyl protease herein. Any fungal polyadenylation sequence, however,is likely to be functional in the present invention.

[0048] A “signal sequence” is an amino acid sequence which when operablylinked to the amino-terminus of a heterologous polypeptide permits thesecretion of such heterologus polypeptide from the host filamentousfungus. Such signal sequences may be the signal sequence normallyassociated with the heterologous polypeptide (i.e., a native signalsequence) or may be derived from other sources (i.e., a foreign signalsequence). Signal sequences are operably linked to a heterologouspolypeptide either by utilizing a native signal sequence or by joining aDNA sequence encoding a foreign signal sequence to a DNA sequenceencoding the heterologous polypeptide in the proper reading frame topermit translation of the signal sequence and heterologous polypeptide.Signal sequences useful in practicing the present invention includesignals derived from bovine preprochymosin (15), A. niger glucoamylase(39), the Mucor miehei carboxyl protease herein and Trichoderma reeseicellulases (29). However, any signal sequence capable of permittingsecretion of a heterologous polypeptide is contemplated by the presentinvention.

[0049] A “propeptide” or “pro sequence” is an amino acid sequencepositioned at the amino terminus of a mature biologically activepolypeptide. When so positioned the resultant polypeptide is called azymogen. Zymogens, generally, are biologically inactive and can beconverted to mature active polypeptides by catalytic or autocatalyticcleavage of the propeptide from the zymogen.

[0050] In one embodiment of the invention a “transformation vector” is aDNA sequence encoding a heterologous polypeptide and a DNA sequenceencoding a heterologous or homologous signal sequence operably linkedthereto. In addition, a transformation vector may include DNA sequencesencoding functional promoter and polyadenylation sequences. Each of theabove transformation vectors may also include sequences encoding anexpressible selection characteristic as well as sequences which increasethe efficiency of fungal transformation.

[0051] “Transformation” is a process wherein a transformation vector isintroduced into a filamentous fungus. The methods of transformation ofthe present invention have resulted in the stable integration of all orpart of the transformation vector into the genome of the filamentousfungus. However, transformation resulting in the maintenance of aself-replicating extra-chromosomal transformation vector is alsocontemplated.

[0052] General Methods

[0053] “Digestion” of DNA refers to catalytic cleavage of the DNA withan enzyme that acts only at certain locations in the DNA. Such enzymesare called restriction enzymes, and the sites for which each is specificis called a restriction site. “Partial” digestion refers to incompletedigestion by a restriction enzyme, i.e., conditions are chosen thatresult in cleavage of some but not all of the sites for a givenrestriction endonuclease in a DNA substrate. The various restrictionenzymes used herein, are commercially available and their reactionconditions, cofactors and other requirements as established by theenzyme suppliers were used. In general, about 1 microgram of plasmid orDNA fragment is used with about 1 unit of enzyme and about 20microliters of buffer solution. Appropriate buffers and substrateamounts with particular restriction enzymes are specified by themanufacturer. Incubation times of about one hour at 37° C. areordinarily used, but may vary in accordance with the supplier'sinstructions. After incubation, protein is removed by extraction withphenol and chloroform, and the digested nucleic acid is recovered fromthe aqueous fraction by precipitation with ethanol. Digestion with arestriction enzyme may be followed by bacterial alkaline phosphatasehydrolysis of the terminal 5′ phosphates to prevent the two ends of aDNA fragment from forming a closed loop that would impede insertion ofanother DNA fragment at the restriction site upon ligation.

[0054] “Recovery” or “isolation” of a given fragment of DNA from arestriction digest means separation of the digest by a polyacrylamidegel electrophoresis, identification of the fragment of interest, removalof the gel section containing the desired fragment, and separation ofthe DNA from the gel generally by electroelution (30).

[0055] “Ligation” refers to the process of forming phosphodiester bondsbetween two double-stranded nucleic acid fragments (30). Unlessotherwise stated, ligation was accomplished using known buffers inconditions with one unit of T4 DNA ligase (“ligase”) per 0.5 microgramof approximately equal molar amounts of the DNA fragments to be ligated.

[0056] “Oligonucleotides” are short length single or double strandedpolydeoxynucleotides which were chemically synthesized by the method ofCrea et al., (31) and then purified on polyacrylamide gels.

[0057] “Transformation” means introducing DNA in to an organism so thatthe DNA is maintained, either as an extrachromosomal element orchromosomal integrant. Unless otherwise stated, the method used hereinfor transformation of E. coli was the CaCl₂ method (30).

[0058]A. nidulans strain G191 (University of Glasgow culture collection)was transformed by incubating A. nidulans sphaeroplasts with thetransformation vector. The genotype of strain G191 is pabaA1 (requiresp-aminobenzoic acid), fwA1 (a color marker), mauA2 (monoaminenon-utilizing), and pyrG89 (deficient for orotidine phosphatedecarboxlase). Sphaeroplasts were prepared by the cellophane method ofBallance et al. (21) with the following modifications. To digest A.nidulans cell walls, Novozyme 234 (Novo Industries, Denmark) was firstpartially purified. A 100 to 500 mg sample of Novozyme 234 was dissolvedin 2.5 ml of 0.6M KCl. The 2.5 ml aliquot was loaded into a PD10 column(Pharmacia-Upsulla, Sweden) equilibrated with 0.6M KCl. The enzymes wereeluted with 3.5 ml of the same buffer.

[0059] Cellophane discs were incubated in Novozyme 234 (5 mg/ml) for 2hours, then washed with 0.6M KCl. The digest and washings were combined,filtered through miracloth (Calbiochem-Behring Corp., La Jolla, Calif.),and washed as described (21). Centrifugations were in 50 or 15 mlconical tubes at ca. 1000×g for 10 min. Following incubation on ice for20 min, 2 ml of the polyethylene glycol 4000 solution (250 mg/ml) wasadded, incubated at room temperature for 5 min. followed by the additionof 4 ml of 0.6M KCl, 50 mM CaCl₂. Transformed protoplasts werecentrifuged, resuspended in 0.6M KCl, 50 mM CaCl₂, and plated asdescribed (21). Zero controls comprised protoplasts incubated with 20 μlof 20 mM Tris-HCl, 1 mM EDTA, pH 7.4 without plasmid DNA. Positivecontrols comprised transformation with 5 μg of pDJB3 constructed asdescribed herein. Regeneration frequencies were determined by platingdilutions on minimal media supplemented with 5-10 ppm paba and 500 ppmuridine. Regeneration ranged from 0.5 to 5%.

[0060] Because of the low transformation frequencies associated withpDJB1, the derivative containing the Mucor acid protease gene (pMeJB1-7)was expected to give extremely low transformation frequencies.Consequently, to obtain pmeJB11-7 transformants of A. nidulans,cotransformation was used. This was accomplished by first constructing anon-selectable vector containing ANS-1, and then transformingsphaeroplasts with a mixture of pmeJB1-7 and the non-selectable vectorcontaining the ANS-1 fragment. The rationale for this approach was thatthe ANS-1 bearing vector would integrate in multiple copies and provideregions of homology for pMeJB1-7 integration. The ANS-1 vector wasprepared by subcloning the PstI-PvuII fragment of ANS-1 (FIG. 12A and13B) from pDJB-3 into pUC16 (33).

[0061] The two plasmids (pMeJB1-7 and the ANS-1 containing vector) weremixed (2.5 μg each) and the above mentioned transformation protocolfollowed.

[0062] Transformants obtained with vectors PGRG1-pGRG4 and pDJB-gam weretransferred after 3 or 4 days incubation at 37° C. Minimal media agarplates supplemented with 5 ppm p-aminobenzoic acid were centrallyinoculated with mycelial transfers from transformants. Three to fivedays following inoculation of minimal medium plates, spore suspensionswere prepared by vortexing a mycelial fragment in 1 ml distilled H₂0,0.02% tween-80. Approximately 5×10⁴ spores were inoculated into 250 mlbaffled flasks containing 50 ml of the following medium: (g/l)Maltodextrin M-040 (Grain Processing Corp., Muscatine, Iowa) 50 g, NaN0₃6 g, MgSO₄.7H20 0.5 g, KCl 0.52 g, KH₂P0₄, 68 g, 1 ml trace elementsolution (34), 1 ml MAZU DF-60P antifoam (Mazer Chemicals, Inc., Gurnee,Ill.), 10 ppm p-aminobenzoic acid, and 50 ppm streptomycin sulfate.Alternatives to MAZU, such as bovine serum albumin or other appropriatesurfactant may be used. Mucor acid protease secretion was tested inAspergillus complete medium (20 g dextrose, 1 g peptone, 20 g maltextract per liter). Carbon source regulation of chymosin secretion byAspergillus nidulans transformants was assessed by measuring secretionin the above-mentioned starch medium relative to the same mediumsupplemented with 1% fructose, sucrose, or dextrose instead of 5%starch. In all cases, the media were incubated at 37° C. on a rotaryshaker (150 rpm). A pDJB3-derived transformant was included as acontrol.

[0063] Western blots of the various secreted chymosins and Mucor mieheicarboxyl protease were performed according to Towbin, et. al (35). Dueto the high concentration of salt in chymosin culture broths and theeffect this salt has on gel electrophoresis a desalting step wasnecessary. Pre-poured G-25 columns (Pharmacia, PD10) were equilibriatedwith 50 mM Na₂HPO₄, pH 6.0. A 2.5 ml aliquot of culture broth wasapplied to the column. The protein was eluted with 3.5 ml of the samebuffer. The heterologous polypeptides present on the blots were detectedby contacting the nitrocellulose blots first with rabbit anti-chymosin(36) or rabbit anti-Mucor miehei carboxy protease serum (36). The blotswere next contacted with goat-anti-rabbit serum conjugated withhorseradish peroxidase (Bio-Rad, Richmond, Calif.) and developed. Priorto loading on the gels, 50 μl of medium (desalted in the case ofchymosin) was mixed with 25 μl of SDS sample buffer. β-mercaptoethanolwas added to a final concetration of 1%. The sample was heated in a 95°C. bath for 5 minutes after which 40-50 μl of sample was loaded on thegel. Each gel was also loaded with 2 μl each of 650, 65 and 6.5 μg/mlchymosin standards and molecular weight markers.

[0064] Western blots of pmeDJ1-7 transformants were similarly analyzedexcept that gel permeation was not performed.

[0065] Protease activity was detected as described by Sokol, et. al.(37). Luria broth was supplemented with 1-1.5% skim milk (Difco) and30-35 ml was poured into a 150 mm petri dish. An aliquot of 2 to 5 μl ofculture medium was spotted on the plate. The plate was incubated overnight at 37° C. in a humidity box. The activity was determined based onthe amount of milk clotting occurring on the plate measured in mm. Theplates were co-spotted with dilutions of 100 CHU/ml or 16.6 CHU/mlrennin (CHU-Chr Hansen Unit, Chr Hansen's Laboratorium, A./S.,Copenhagen). The relationship between the diameter of the coagulationzone (mm) and the centration of enzyme is logarithmic.

[0066] In order to distinguish between types of proteases, pepstatin, aninhibitor of the chymosin type of carboxyl protease, was used to inhibitprotease activity attributable to chymosin. Samples of chymosin mutantsand control broths were preincubated with a 1:100 dilution of 10 mMpepstatin in DMSO for 5 minutes before analyzing for protease activity.

[0067] Glucoamylase secretion by pDJB-gam-l transformants in 5% starchmedia was assessed using an assay based on the ability of glucoamylaseto catalyze the conversion of p-nitrophenol-a-glucopyranoside (PNPAG)(38) to free glucose and p-nitrophenoxide. The substrate, PNPAG, wasdissolved in DMSO at 150 mg/ml and 3 to 15 μl aliquots were diluted to200 ul with 0.2 M sodium acetate, 1 mM calcium chloride at pH 4.3. A 25μl sample was placed into a microtitre plate well. An equal volume ofstandards ranging from 0 to 10 Sigma A. niger units/ml (Sigma ChemicalCo., St. Louis, Mo.) were placed in separate wells. To each well, 200 μlof PNPAG solution at 2.25 to 11.25 mg/ml was added. The reaction wasallowed to proceed at 60° C. for 0.5 to 1 hour. The time depended uponthe concentration of enzyme. The reaction was terminated by the additionof 50 μl of 2 M trizma base. The plate was read at 405 nm. Theconcentration of enzyme was calculated from a standard curve.

[0068] Unless otherwise stated, chromosomal DNA was extracted fromfilamentous fungi by the following procedure. The filamentous fungus wasgrown in an appropriate medium broth for 3 to 4 days. Mycelia wereharvested by filtering the culture through fine cheesecloth. The myceliawere rinsed thoroughly in a buffer of 50 mM tris-HCl, pH 7.5, 5 mM EDTA.Excess liquid was removed by squeezing the mycelia in the cheesecloth.About 3 to 5 grams of wet mycelia were combined with an equivalentamount of sterile, acid-washed sand in a mortar and pestle. The mixturewas ground for five minutes to form a fine paste. The mixture was groundfor another five minutes after adding 10 ml of 50 mM tris-HCl, pH 7.5, 5mM EDTA. The slurry was poured into a 50 ml capped centrifuge tube andextracted with 25 ml of phenol-chloroform (equilibrated with an equalvolume of 50 mM tris-HCl, pH 7.5, 5 mM EDTA). The phases were separatedby low speed centrifugation. The aqueous phase was saved and reextractedthree times. The aqueous phases were combined (about 20 ml total volume)and mixed with 2 ml of 3 M sodium acetate, pH 5.4 in sterile centrifugetubes. Ice cold isopropanol (25 ml) was added and the tubes were placedat −20° C. for one hour. The tubes were then centrifuged at high speedto pellet the nucleic acids, and the supernatant fluid was discarded.Pellets were allowed to air dry for 30 minutes before resuspending in400 μl of 10 mM tris-HCl, pH 7.5, 1 mM EDTA (TE buffer). Pancreaticribonuclease (Sigma Chemical Co., St. Louis, Mo.) was added to a finalconcentration of 10 μg per ml, and the tubes were incubated for 30minutes at room temperature (30). Ribonuclease was then removed byextraction with phenol-chloroform. The aqueous layer was carefullyremoved and placed in a tube which contained 40 μl of 3M sodium acetate,pH 5.4. Ice cold ethanol was layered into the solution. The DNAprecipitated at the interface and was spooled onto a glass rod. This DNAwas dried and resuspended in a small volume (100 to 200 μl) of TEbuffer. The concentration of DNA was determined spectrophotometricallyat 260 nm (30).

[0069] To confirm the chromosomal integration of chymosin DNA sequencesin selected transformants Southern hybridizations were performed (30).Spore suspensions of transformants were inoculated into Aspergilluscomplete medium and incubated at 37° C. on a rotary shaker for 24-48hrs. The medium was non-selective in that it was supplemented with 5 ppmp-aminobenzoic acid and contained sufficient uracil for growth of theauxotrophic parent. In effect, these Southerns also tested for thestability of the transformants. The mycelium was filtered, ground insand, and the DNA purified as previously described. Transformant DNA wasthen digested with various restriction enzymes and fragments separatedby agarose gel electrophoresis. Control lanes included digested pDJB3transformant DNA and undigested DNA. Gels were stained with ethidiumbromide, photographed, blotted to nitrocellulose or nytran (Schleicherand Schuell, Keene, N.H.), and probed with radiolabeled plasmids orspecific fragments.

EXAMPLE 1

[0070] Expression and Secretion of Aspergillus niger glucoamylase ByAspergillus Nidulans

[0071] A. Construction of pGA1

[0072] Aspergillus niger (Culture #7, Culture Collection Genencor, Inc.,South San Francisco, Calif.) was grown in potato dextrose broth (Difco,Detroit, Mich.) at 30° C. for 3 days with vigorous aeration. ChromosomalDNA was extracted as previously described.

[0073] A synthetic oligonucleotide was used as a hybridization probe todetect the glucoamylase gene from Aspergillus niger. The oligonucleotidewas 28 bases in length (28 mer) and corresponded to the first 9⅓ codonsof the published glucoamylase coding sequence (39):MetSerPheArgSerLeuLeuAlaLeuSer 5′ATGTCGTTCCGATCTCTACTCGCCCTGA3′

[0074] The oligonucleotide was synthesized on a Biosearch automated DNAsynthesizer (Biosearch, San Rafael, Calif.) using the reagents andprotocols specified by the manufacturer.

[0075] Genomic DNA from Aspergillus niger was analyzed for the presenceof glucoamylase sequences by the method of Southern (30). Briefly, 10 μgof Aspergillus niger DNA was digested with EcoRl restrictionendonuclease. The digested DNA was subjected to electrophoresis on a 1%agarose gel according to standard methods (30). DNA was transferred fromthe gel to a nitrocellulose membrane (Schleicher & Schuell, Inc., Keene,N.H.) by blotting in 10×SSC (1.5 M NaCl, 0.15 M trisodium citrate) (30).DNA was fixed to the nitrocellulose by baking in an 80° C. vacuum oven,followed by hybridization at low stringency (2,40) with radiolabeledoligonucleotide probe. Radiolabeling of the synthetic oligonucleotidewas done at 37° C. in a 50 μl reaction that contained 70 mM tris-HCl, pH7.5, 10 mM MgCl₂, 5 mM dithiothreitol, 30 pmoles of syntheticoligonucleotide, 20 pmoles of gamma-[32P]ATP (Amersham, Chicago, Ill.;specific activity 5000 Ci/mmol), and 5 units of T4 polynucleotide kinase(New England Biolabs). After hybridization, the filters were washed 15minutes in 2×SSC, 0.1% sodium dodecylsulfate (SDS) and twice in 2×SSC at37° C. Filters were air dried, wrapped in Saran-Wrap (Dow Chemical) andapplied to Kodak X0mat-AR X-ray film at −70° C. to obtain anautoradiographic image. After developing the autoradiogram, a band ofhybridization was clearly visible corresponding to a 3.5 kilobase-pairEcoRl fragment.

[0076] Genomic DNA from Aspergillus niger was digested with EcoRl andsize-fractionated by polyacrylamide gel electrophoresis according tostandard methods (30). DNA fragments 3 to 4 kb in size were excised andeluted from the gel (30). This DNA fraction was used to generate alibrary of clones in the Escherichia coli cloning vector pBR322 (ATCC37017). The cloning vector was cleaved with EcoRl and dephosphorylatedwith bacterial alkaline phosphatase (Bethesda Research Labs). A typicaldephosphorylation reaction consisted of 1 μg of digested vector DNA and1 unit of alkaline phosphatase in 50 μl of 50 mM tris-HCl, pH 8.0, 50 mMNaCl. The reaction was incubated at 65° C. for one hour. The phosphatasewas removed by extraction with phenol-chloroform. The EcoRl,size-selected Aspergillus niger DNA was then ligated with EcoRl cleavedand dephosphorylated pBR322. A typical ligation reaction contained thefollowing: 100 ng each of vector and insert DNAs, 1 unit of T4 DNAligase (Bethesda Research Labs), 25 mM tris-HCl, pH 7.5, 10 mM MgCl₂, 10mM dithiothreitol, and 1 mM ATP in a 10 μl volume. Ligation reactionswere incubated at 16° C. for 18 to 24 hours. The ligated DNA was used totransform competent A. coli. 294 cells (ATCC 31446) prepared by themethod of Morrison (41). Transformants were selected on LB agar plates(30) which contained carbenecillin at a final concentration of 50 μg perml. Transformants which harbored glucoamylase gene sequences wereidentified by colony hybridization methods (30) using theglucoamylase-specific 28 mer as a probe. Hybridizing colonies werepurified, and plasmid DNAs were isolated from each by the alkaline-SDSminiscreen procedure (30). The plasmids selected in this manner allcontained a 3.5 kb EcoRl fragment which hybridized to the syntheticglucoamylase probe. One such plasmid, designated pGa1, was selected forfurther analysis. A 1.1 kb EcoRl-BglII fragment from the insert in pGa1was subcloned into M13 mp9 (42) and partially sequenced by the dideoxychain termination method (43) to confirm that the cloned DNA encoded theglucoamylase gene. A restriction endonuclease cleavage map of the 3.5 kbEcoRl fragment contained in pGa1 is depicted in FIG. 1. It was generatedby single and double restriction digests followed by orientation of theDNA fragments with respect to known restriction sites in pBR322 (44).

[0077] B. Construction of pGa5

[0078] The nucleotide sequence and restriction map of pGa1 indicatedthat pGa1 contained the entire glucoamylase coding region and 221nucleotides of 5′ flanking DNA. The sequences in this 5′ region werestrikingly similar to typical eukaryotic promoter sequences with TATAAATand CAAT boxes located upstream of the ATG start codon (48).

[0079] However, to insure that possible upstream activation sites of theAspergillus niger glucoamylase gene were included in the finaltransformation vector a larger genomic fragment which contained at least1000 bp of 5′ flanking DNA was cloned. Southern blotting experimentssimilar to those already described identified a 6.5 kb ClaI fragmentwhich hybridized to a radiolabeled EcoRI glucoamylase fragment frompGa1. The EcoRI fragment was radiolabeled by nick translation (30) withalpha-[32P]dCTP (Amersham; specific activity 3000 Ci/mmol). A nicktranslation kit (Bethesda Research Labs) was used for the labelingreaction by following the instructions supplied by the manufacturer.Filters were hybridized and washed under stringent conditions (30).

[0080] The 6.5 kb ClaI fragment identified by hybridization was clonedin a manner similar to that described previously. Aspergillus nicer DNAwas digested with ClaI and size-fractionated by polyacrylamide gelelectrophoresis. DNA fragments migrating between 5.5 and 8 kb wereexcised and eluted from the gel. This fraction was ligated to ClaIcleaved and dephosporylated pBR325 (45). The ligation mixture was usedto transform competent E. coli 294 cells. Transformants were selected onLB agar plates containing carbenecillin (50 μg/ml). Colonies whichcontained glucoamylase gene sequences were identified by colonyhybridization (30). Plasmid DNA extracted from hybridizing coloniescontained a 6.5 kb ClaI fragment which included the 3.5 kb EcoRlfragment cloned previously in pGa1. These recombinant plasmids encodedthe Aspergillus niger glucoamylase gene as confirmed by supercoil-DNAsequencing (46) with the synthetic oligonucleotide (28 mer) as asequencing primer. A restriction endonuclease cleavage map of the 6.5 kbClaI fragment was constructed using single and double digests of the DNAcloned in pBR325. Restriction sites in the vector DNA were used asreference points to orient the fragment. This restriction map is shownin FIG. 1. Location of the, glucoamylase gene was deduced by comparingrestriction sites of pGa5 to those of the previously publishedglucoamylase genes (39, 47, 48). From the mapping data it was estimatedthat approximately 3.3 kb of the 5′-flanking DNA and about 1 kb of3′-flanking DNA were contained within the cloned fragment.

[0081] Plasmid pGa5 was deposited with the ATCC on Aug. 28, 1985 in E.coli 294 and has been assigned number 53249.

[0082] C. Vector for Expression and Secretion of Aspergillus nigerGlucoamylase

[0083] The 6.5 kb ClaI fragment from pGa5 containing the glucoamylasegene was cloned into the E. coli.-Aspergillus nidulans shuttle vectorpDJB3 as depicted in FIG. 2. The pDJB3 shuttle vector possesses aselectable beta-lactamase gene and origin or replication from E. coliplasmid pBR325, the pyr4 gene from Neuospora crassa which relieves theauxotrophic requirement for uridine in Aspergillus nidulans strain G191,a sequence known as ANS1 from Aspergillus nidulans which promotes a highfrequency of stable integrative transformants in Aspergillus nidulans,unique EcoRl and ClaI restriction sites for cloning.

[0084] pDJB is constructed as depicted in FIG. 14. Plasmid pFB6 (32) isdigested to completion with BglII and partially digested with HindIII.Fragment B containing the pyr4 gene (ca. 2 Kb) is purified by gelelectrophoresis and ligated into HindIII/Bam HI digested pBR325(fragment A) yielding plasmid pDJB1. The ANS-1 sequence is cloned byligating EcoRI digested A. nidulans genomic DNA (strain G191 or otherFGSC#4-derived strains) into EcoRl cleaved pFB6. The resulting pool ofEcoRI fragments in pFB6 is used to transform a ura3-S. cerevisiae (E.G.ATCC 44769, 44770 etc.). An autonomously replicating plasmid, pIntA, ispurified from the S. cerevisiae transformant. pIntA is digested withEcoRI, the ANS-1 fragment is purified by gel electrophoresis and ligatedinto EcoRI digested pDJB1, yielding plasmid pDJB2. pDJB2 is partiallydigested with EcoRI, treated with DNA polymerase I (Klenow), andre-ligated to yield plasmid pDJB3. The partial nucleotide sequence andrestriction map of the ANS-1 fragment is showin in FIG. 13A and 13B.

[0085] Plasmid pGa5 was digested with ClaI and the large fragment(fragment A) was separated from the vector by agarose gelelectrophoresis. This fragment was ligated with pDJB3 which had beencleaved with ClaI and dephosphorylated (fragment B). The ligationmixture was used to transform competent E. coli 294 cells. Transformantswere selected on LB agar supplemented with carbenecillin (50 μg/ml).Analysis of plasmid DNAs from these transformants indicated that theglucoamylase fragment had been inserted as expected. Both orientationsof the glucoamylase fragment were obtained by screening varioustransformants. One plasmid, designated pDJB-gam1 was arbitrarily chosenfor transformation of Aspergillus nidulans protoplasts.

[0086] D. Expression and Secretion of Glucoamylase

[0087]Aspergillus nidulans Strain G191 was transformed with pDJB-gam-1as previously described. Five transformants designated pDJB-gam-1-4, 9,10, 11 & 13 were analyzed for glucoamylase activity as previouslydescribed. The results are shown in Table I. TABLE I GlucoamylaseActivity Sample (Sigma Units/ml) pDJB3 0.129 pDJB-gam-1-4 0.684pDJB-gam-1-9 0.662 pDJB-gam-1-10 0.131 pDJB-gam-1-11 0.509 pDJB-gam-1-130.565 A. niger 2.698

[0088] As can be seen, each pDJB-gam-1 transformant produced moreglucoamylase activity than the control indicating that biologicallyactive glucoamylase was expressed and secreted from the transformedfungi.

EXAMPLE 2

[0089] Expression and Secretion of Bovine Chymosin from Aspergillusnidulans

[0090] Expression vectors were constructed encoding either a naturalprecursor of bovine chymosin (preprochymosin) or a fusion precursor inwhich DNA sequences for Aspergillus niger glucoamylase and prochymosinwere precisely fused. The strategy for the construction of these vectorsinvolved the following steps. First, a DNA sequence containing a portionof the glucoamylase promoter and a portion of the glucoamylase 5′-codingregion was cloned upstream from a DNA sequence corresponding to theamino-terminal portion of preprochymosin. Next, nucleotides between theDNA fragments were deleted by M13 site-specific mutagenesis (40) usingspecific primer sequences. Finally, a segment of DNA containing thefused sequences was incorporated with the remaining portion of theprochymosin sequence into an expression vector which employed the 5′-and 3′-regulatory sequences of the Aspergillus niger glucoamylase gene.These steps are outlined in FIGS. 3 through 7.

[0091] A. Construction of mp19 GAPR

[0092] Plasmid pGa5 is used to derive a 337 bp EcoRl-RsaI DNA fragment(fragment A) bearing a portion of the glucoamylase promoter and anamino-terminal segment of the coding region. Fragment A was ligated withEcoRl and SmaI digested M13 mp19 RF-DNA (fragment B). The ligationmixture was used to transform E. coli. JM101 (ATCC 33876). Clear plaqueswere analyzed for the presence of fragment A by restriction analysis ofthe corresponding RF-DNA. One isolate containing fragment A, designatedmp19R-Rsa was digested with PstI and XbaI and the large fragment(fragment C) was isolated. A small XbaI-PstI sequence (fragment D)derived from pR3 (49) containing 5′ preprochymosin sequences; waspurified by electrophoresis and ligated to fragment C to produce thephage template mp19GAPR as shown in FIG. 3.

[0093] B. Site Specific Deletion Mutagenesis

[0094] As shown in FIG. 8 mp19GAPRΔC1 was derived from mp19 GAPR bydeleting the nucleotides between the glucoamylase signal peptide codonsand the codons for prochymosin by site-specific mutagenesis. Thus, inmp19GAPRΔC1 the glucoamylase signal peptide codons are precisely fusedto the first codon of prochymosin. Site-specific mutagenesis was done aspreviously described (40) except that only one oligonucleotide was usedto prime second strand synthesis on the single-stranded M13 template(FIG. 4) (40). The synthetic oligonucleotide used to derive mp19GAPRΔC1(primer 1) was 5′ GCTCGGGGTTGGCAGCTGAGATCACCAG 3′. Plaques containingthe desired deletion were identified by hybridization with the primerradio-labeled as previously described.

[0095] In mp19GAPRΔC3 the nucleotides between those immediatelypreceding the initiation codon of glucoamylase and the ATG start codonof preprochymosin were joined by site-specific mutagenesis using thesynthetic oligonucleotide (primer 3)

[0096] 5′ ACTCCCCCACCGCAATGAGGTGTCTCGT 3′.

[0097] The resulting mutation linked the glucoamylase promoter regionprecisely to the initiation codon of preprochymosin as depicted in FIG.8.

[0098] C. Construction of Vectors for the Expression and Secretion ofBovine Chymosin

[0099] As further depicted in FIG. 4 each of the fusions between theglucoamylase sequences and 5′ prochymosin sequences (m19GAPRΔC1 andmp19GAPRΔC3) were combined with the 3′ prochymosin sequences and theSaccharomyces cerevisiae phosphoglyceratekinase (PGK) terminator in theAspergillus nidulans transformation vector pDJB3. The replicative formof mp19GAPRΔC1 and mp19GAPRΔC3 was digested with EcoRl and PstI. Thesmaller fragment (fragment 1) was isolated. Plasmid pBR322 was alsodigested with EcoRl and PstI and the larger vector fragment (fragment 2)was isolated. Fragments 1 and 2 were joined by ligation and used totransform E. coli. 294. A tetracycline resistant colony containingeither plasmid pBR322GAPRΔC1, or pBR322GAPRΔC3 was isolated. Fragment 2was also treated with E. coli. polymerase I (Klenow fragment). Theresulting blunt ended fragment was circularized by ligation and used totransform E. coli 294. One tetracycline resistant colony containingplasmid pBR322ΔRP was isolated and then digested with HindIII and SalI.The larger vector fragment (fragment 3) was isolated. The plasmid pCR160was digested with HindIII and PstI and fragment 5 (containing the yeastPGK terminator fused to 3′ prochymosin codons) was isolated. Fragments3, 4, and 5 were joined by ligation and used to transform E. coli 294. Atetracycline resistant transformant containing plasmid pBR322GAPR^(Δ)C1or pBR322GAPR^(Δ)C3 was isolated.

[0100] Plasmid pCR160 contains the yeast 2 μm origin of replication toallow its maintenance as a plasmid in yeast, the yeast TRP-1 gene as ayeast selection marker, an E. coli origin or replication and ampicillinresistance gene from the plasmid pBR322, and a prorennin expressionunit. The prorennin expression unit contained the promoter from theyeast PGK gene, the prorennin coding region, and the terminator from thePGK gene. Construction of this plasmid was accomplished as depicted inFIG. 9 in the following manner: Plasmid YEpIPT (50) was partiallydigested with HindIII followed by a complete EcoRl digestion, and thevector fragment A was isolated. A second plasmid pPGK-1600 (51) waspartially digested with both EcoRl and HindIII, and the PGK promoterfragment B was isolated. Fragments A and B were ligated to give theintermediate pInt1 which was again partially digested with EcoRl and theHindIII, and the vector fragment C was isolated. The PGK terminatorfragment D was isolated following HindIII and Sau3A digestion of theplasmid pB1 (52). The prorennin fragment E was isolated by cleaving pR1(49) DNA with EcoRl and BclI. Fragments C, D, and E were then ligated toproduce the yeast expression plasmid pCR160. The nucleotide sequence ofthe PGK promoter, structural gene and terminator have been reported(53).

[0101] Plasmids pBR322GAPRΔC1 and pBR322GAPRΔC3 contain a completetranscriptional unit for each of the forms of prochymosin. Thistranscriptional unit contains a precursor prochymosin coding sequence,the glucoamylase promoter, and the yeast PGK terminator. However,derivatives of these plasmids and plasmids pBR322GAPRΔC2 andpBR322GAPRΔ4, described hereinafter, [designated pint1 (1-4) FIG. 5)]produced no detectable chymosin when used to transform A. nidulans G191.It is not understood why these derivative plasmids failed to express andsecrete chymosin. However, in light of subsequent results it appearsthat the yeast PKG terminator and/or the short glucoamylase promotorsequence in these plasmids is not recognized by A. nidulans G191. Basedon these results, the pBR322GAPRΔC plasmids were further modified.

[0102] In the following steps the transcriptional unit was moved ontothe Aspergillus nidulans transformation vector pDJB3. Additionalglucoamylase 5′ flanking sequences were incorporated just 5′ of thepromoter to insure the presence of possible upstream activation siteswhich could be involved in regulating expression. Further, the PKGterminator was replaced with the A. niger glucoamylase terminator frompGa5. Specifically, in FIG. 5 each plasmid (pBR322GAPRΔC1 orpBR322GAPRΔC2) was digested with ClaI and fragment 6 was isolated.Plasmid pDJB3 was also digested with ClaI and treated with bacterialalkaline phosphatase in order to minimize self-ligation. This digestedplasmid (fragment 7) was joined to fragment 6 and one ampicillinresistant colony containing plasmid pINTI-1 or pIntI-3 was isolated.These plasmids were digested with XhoI and NsiI and the larger vectorfragment (fragment 8 ) was isolated. Plasmid pGa5 which contains theentire glucoamylase gene as well as extensive 5′ and 3′ flankingsequences was digested with XhoI and NsiI and the smaller fragment(fragment 9, containing approximately 1700 bp of these 5′ sequences) wasisolated. Fragments 8 and 9 were joined by ligation and used totransform E. coli 294. One ampicillin resistant colony containingplasmid pInt2-1 or pInt2-3. was isolated. These plasmids differ mostsignificantly from the final vectors (see FIG. 7) in that they containthe yeast PGK terminator rather than the glucoamylase terminator.

[0103] Additional steps in the construction of chymosin expressionvectors are outlined in FIG. 6. Plasmid pR1 (49) was used to isolate asmall BclI-Asp718 DNA fragment (fragment A) which comprised the 3′- endof prochymosin cDNA. Fragment A was subsequently cloned into pUC18 (33)that was digested with Asp718 and BamHI (fragment B). Similarly, a 1.2kb ClaI-Asp718 DNA fragment (fragment D) was isolated from plasmid pGa5,and cloned into AccI and Asp718 cleaved pUC18 (fragment C). Theresulting intermediate plasmids, pUC-int1 and pUC-int2, were digestedwith SalI and HindIII, and fragments E and F were isolated. Thesefragments were then ligated to produce pUC-int3 which contained the 3′end of prochymosin followed by the glucoamylase terminator sequences ona HindIII-Asp718 fragment (fragment H).

[0104] A new cloning vector, designated pBR-link, was created byinserting a synthetic oligonucleotide linker (containing XhoI and ClaIsites) into the unique BamHI site of pBR322. This linker connoted thefollowing sequence: 5′ GATCCATCGATCTCGAGATCGATC 3′3′ GTAGCTAGAGCTCTAGCTACCTAG 5′

[0105] The larger HindIII-XhoI fragment of this vector (fragment G) waspurified by electrophoresis. Similarly, the XhoI-Asp718 restrictionfragments (fragments I) of plasmids pInt2-1 and pInt2-3 were isolatedelectrophoretically. Fragments G and H were ligated with each of thedifferent I-fragments in a series of three-way ligations to produce theintermediates pInt3-1 and pInt3-3. These key intermediates contained theglucoamylase promoter regions, various signal and propeptide fusions tothe prochymosin (or preprochymosin) sequences followed by theglucoamylase terminator region all within convenient ClaI restrictionsites. Because certain ClaI sites, such as those in the linker ofpBR-link, are inhibited by E. coli. DNA methylation, the plasmidspInt3-1 through pInt3-4 were transformed into a dam-strain of E. coli,designated GM48, (ATCC 39099) from which the plasmids were re-isolated.The unmethylated DNA was digested with ClaI and fragment J was purifiedby electrophoresis. Fragment J from each of theglucoamylase-pro-chymosin fusions was subsequently cloned into theunique ClaI site of pDJB3 (fragment K) to produce the final expressionvectors pGRG1 and pGRG3.

[0106] D. Expression and Secretion of Bovine Chymosin

[0107]Aspergillus nidulans G191 was transformed with pGRG1 and pGRG3 aspreviously described.

[0108] Five pGRG1 and five pGRG3 transformants were analyzed. Westernanalysis (not shown) indicated that each transformant secreted a proteinwhich reacted with anti-chymosin and which migrated at the same orslightly higher molecular weight of bovine chymosin. The highermolecular weight species may be due to incorrect processing, mediaeffects, or glycosylation. Integration was confirmed for onetransformant of pGRG3 by Southern hybridization (results not shown).Each transformant was also assayed for chymosin activity. The results ofthis assay are shown in Table II. TABLE II No. of Range Transformants ofChymosin Transformant Tested Activity μg/ml pDJB3 1   0-0.13 pGRG1 5  0-1.5 pGRG3 5 0.05-7.0 

[0109] These results indicate that pGRG2 and pGRG3 both secrete aprotease, at various levels, above the pDJB3 control. Occasionally,background proteolytic activity was detected in pDJB3 control broths. Aswill be shown hereinafter this protease activity of transformants isassociated with the aspartic acid family of carboxyl proteases of whichchymosin is a member.

EXAMPLE 3

[0110] Expression and Secretion of Fusion Polypeptides from AspergillusNidulans

[0111] Two fusion polypeptides were constructed for expression andsecretion from A. nidulans. One fusion polypeptide contained anamino-terminal portion consisting of the pro sequence and first tenamino acids of Aspergillus niger glucoamylase and a carboxyl-terminalportion consisting of bovine prochymosin. The second fusion polypeptidecontained an amino-terminal portion consisting of the pro sequence onlyof Aspergillus niger glucoamylase and a carboxyl-terminal portionconsisting of bovine prochymosin.

[0112] A. Vectors for Expressing and Secreting Fusion Polypeptides

[0113] Vectors encoding the above fusion polypeptides were constructedby deleting specific sequences from mp19GAPR followed by the samemanipulations as described above for constructing pGRG1 and pGRG3. Asshown in FIG. 8, in mp19GAPRΔC2 the nucleotides between the glucoamylasepropeptide codons and the codons of prochymosin were deleted using thesite-specific mutagenesis method described above. The sequence of theoligonucleotide synthesized for this mutagenesis (primer 2) was

[0114] 5′ TGATTTCCAAGCGCGCTGAGATCACCAG 3′.

[0115] This mutation was intended to fuse the glucoamylase promoter,signal peptide, and propeptide codons to the first codon of prochymosin.In mp19GAPRΔC4 the nucleotide sequences between the tenth codon ofmature glucoamylase and the codons of prochymosin were deleted by M13site-specific mutagenesis with the synthetic oligonucleotide (primer 4)

[0116] 5′ TGAGCAACGAAGCGGCTGAGATCACCAG 3′.

[0117] This deletion fused the glucoamylase promoter region, signalpeptide sequence, propeptide sequence, plus ten codons of the matureglucoamylase to the codons of prochymosin as shown in FIG. 8. Theseexpression and secretion vectors designated as pGRG2 and pGRG4 were usedto transform A. nidulans.

[0118] B. Expression and Secretion of Chymosin from Aspergillus NidulansTransformed With PGRG2 and PGRG4

[0119] pGRG2 and pGRG4 transformants were cultured as previouslydescribed. The culture medium was assayed for chymosin activity byWestern blot and gave results similar to those obtained for pGRG1 andpGRG3. Integration of one pGRG2 transformant was confirmed by SouthernAnalysis (results not shown). The results of the chymosin assay arepresented in Table III. TABLE III No. of Range Transformants of ChymosinTransformant Tested Activity μg/ml pDJB3 1    0-0.13 pGRG2 1 0.001-0.42pGRG4 6 0.004-0.75

[0120] Again each of the transformants demonstrated protease activityabove the pDJB3 control indicating that a protease was expressed andsecreted by the transformants. As with pGRG1 and pGRG3, these proteasesbelong to the aspartic acid family of carboxyl proteases as evidenced bythe pepstatin inhibition. Significantly, these results indicate thathybrid polypeptides have been expressed in a filamentous fungus.

EXAMPLE 4

[0121] Pepstatin Inhibition Study

[0122] Three of the above vectors containing the various constructionsinvolving chymosin were analyzed in the pepstatin inhibition assay asdescribed supra. The results are shown in Table IV. TABLE IV ChymosinSample Activity (μg/ml) pDJB3 0 pDJB3 pepstatin 0 pGRG1 0.2 pGRG1pepstatin 0.05 pGRG2 0.1 pGRG2 pepstatin 0 pGRG3 3 pGRG3 pepstatin 0.6

[0123] The samples preincubated with pepstatin show a marked decrease inactivity indicating that the protease produced by the transformants isof the aspartic acid family of acid proteases to whic chymosin is amember. This data together with the results from the Western analysisindicates that biologically active chymosin is expressed and secreted byA. nidulans G191 transformed with pGRG1, pGRG2, pGRG3 and pGRG4.

[0124] The variation in the amount of chymosin activity detected fordifferent vector constructions in Example II and Example III may reflectdifferences in the recognition of the various signals incorporated ineach transformation vector. Within a particular construction, thevariation in chymosin activity may be related to the copy number of thevector incorporated into the fungal genome and/or to the location ofsuch integration.

EXAMPLE 5

[0125] Carbon Source Studies

[0126] One vector, pGRG4, was used to transform A. nidulans G191 whichwas thereafter grown on the various carbon sources previously described.The results of this assay are shown in Table V.

[0127] Table V

[0128] Amount of chymosin activity produced on various carbon sources(^(μ)g/ml) starch glucose fructose sucrose pDJB3 0   0   0   0   pGRG43.5 3.5 0.9 1.75

[0129] These results clearly show that chymosin is secreted regardlessof the carbon source. This suggests that transcriptional regulation ofthe glucoamylase promotor is unlike that in A. niger, i.e. not stronglyinducible by starch.

EXAMPLE 6

[0130] Expression and Secretion of Mucor meihei Carboxyl Protease

[0131] A. Carboxyl Protease Genomic Probe

[0132] The partial primary structure of Mucor miehei acid protease (54)was inspected for the region of lowest genetic redundancy. Residues187-191 (using the pig pepsin numbering system), try-tyr-phe-trp-asp,were selected. Oligonucleotides complementary to the coding sequencecorresponding to this amino acid sequence,

[0133] 5′-GC(G/A)TCCCA(G/A)AA(G/A)TA(G/A)TA-3′,

[0134] were synthesized (31) and labelled using gamma 32P-ATP and T4polynucleotide kinase (30).

[0135] B. Cloning of Mucor meihei Carboxyl Protease

[0136] Genomic DNA from Mucor miehei (Centraal Bureau VoorSchimmelcultures, Holland 370.75) was prepared as follows. Cells grownin YMB medium (3 g/l yeast extract, 3 g/l malt extract, 5 g/l peptone,10 g/l glucose) were collected by centrifugation, washed twice with 0.5MNaCl, and lyophilized. Cell walls were then disrupted by adding sand tothe cells and grinding the mixture with a mortar and pestle. Theresulting powder was suspended (15 ml. per gram dry weight) in asolution containing 25% sucrose, 50 mM Tris-HCl (pH 8.0), and 10 mMEDTA. SDS was added to a final concentration of 0.1% and the suspensionwas extracted once with a half-volume of phenol and three times withhalf volumes of chloroform. The final aqueous phase was dialysedextensively against 10 mM Tris-HCl, pH 8.0 and 1 mM EDTA. The DNA wasthen precipitated by the addition of sodium acetate, pH 5.5, to aconcentration of 0.3 M. followed by the addition of 2.5 volumes of coldethanol. Aliquots of this DNA were digested with a variety ofrestriction endonucleases according to the manufacturers' directions andthen analyzed for sequences complementary to sequences of the probesdescribed above, using the method of Southern. A positively hybridizingband of approximately 2.5 kb (kilobases) was identified in the HindIIIdigested DNA. HindIII digested genomic DNA was separated bypolyacrylamide gel electrophoresis and a gel fragment containing DNA of2.0-3.0 kb was electroeluted as previously described. The electroelutedDNA, presumed to be enriched for sequences corresponding to the Mucormiehei acid protease gene, was ethanol precipitated. The cloning vectorpBR322 (ATCC 37017) was digested with HindIII and dephosphorylated usingbacterial alkaline phosphatase. In a typical 10 ul reaction 100 ng ofvector and 100 mg of the size enriched DNA were joined in the presenceof ATP and T4 DNA ligase. The reaction was used to transform E. coli 294(ATCC 31446) by the calcium shock procedure (30). About 2.0×10⁴ampicillin resistant clones were obtained. Approximately 98% of thesecontained cloned inserts as indicated by their failure to grown ontetracycline containing medium. These colonies were tested by a standardcolony hybridization procedure for the presence of sequencescomplementary to those of the DNA probes. One positively hybridizingcolony, containing plasmid pMe5′muc, was found to contain a HindIIIinsert of the expected 2.5 kb size. The termini of this fragment weresubcloned into M13 sequencing vectors (33) and their sequencesdetermined by the dideoxy chain termination method. One terminuscontained sequences corresponding to the known amino terminal amino acidsequence of the acid protease gene. The adjacent 3′ region was sequencedin order to obtain more C terminal coding sequences. The sequencingstrategy is shown in FIG. 10. In this way the entire coding sequence forthe mature form of the protein was obtained. The 5′ end of the fragmentwas found to occur 112 bp (base pairs) upstream of the codoncorresponding the mature amino terminus. Since this upstream regioncontained no in frame initiation codons it was presumed to be part of apropeptide.

[0137] In order to obtain DNA containing the initiation codon as well as5′ untranslated sequences a more 5′ clone was isolated as follows. AHindIII-ClaI 813 bP 5′ subfragment of the pMe5′Cla Hind III insert wasisolated and labelled by the nick translation method (30). This labelledfragment was used to probe ClaI digested Mucor miehei genomic DNA by themethod of Southern. This experiment revealed a single band ofhybridization corresponding to a molecular weight of approximately 1300bp. Size enriched DNA of this size was isolated and cloned into ClaIdigested and dephosphorylated pBR322 as described above.

[0138] Approximately 9000 ampicillin resistant colonies were obtained.About 90% of these contained cloned inserts as indicated by theirfailure to grow on tetracycline containing medium. These colonies weretested by a standard colony hybridization procedure for the presence ofsequences complementary to those of the nick translated probe. Onepositively hybridizing colony, containing plasmid pMe2, was found tocontain a ClaI insert of the expected 1.3 kb size. Sequencing of theends of this fragment showed that one terminus corresponded to sequencesnear the ClaI site of the Hind III fragment in pMe5′Cla and thuspermitted orientation of the fragment which is shown in FIG. 10. Furthersequencing of the ClaI fragment disclosed the initiation codon and 5′untranslated sequences. The entire coding sequence and the 5′ and 3′flanking sequences are shown in FIG. 10. Comparison of the deducedprimary structure with that determined by direct amino acid sequencingindicates that the Mucor protein is made as a precursor with anamino-terminal extension of 69 residues. Based on the structuralfeatures generally present in leader peptides it is likely that residues−21 to −1 comprise a leader peptide and that residues 21-69 comprise apropeptide analogous to that found in the zymogen forms of other acidproteases including chymosin and pepsin (55).

[0139] C. Mucor meihei carboxyl protease Expression and Secretion Vector

[0140] A vector for expressing and secreting Mucor miehei carboxyprotease includes the entire native Mucor miehei acid proteasetranscriptional unit including the coding sequence, 5′ flankingsequences (promoter), and 3′ flanking sequences (terminator andpolyadenylation site).

[0141] The overall strategy for making this vector is depicted in FIG.12. The Aspergillus nidulans transformation vector pDJB1 was digestedwith ClaI and EcoRl and the larger vector fragment (fragment 1) wasisolated. The plasmid pMe5′Cla was digested with EcoRl and ClaI andfragment 2 was isolated. This fragment contains the 5′ codons of theacid protease together with about 500 bp of 5′ flanking sequences.Fragments 1 and 2 were joined by ligation and used to transform E. coli294. One ampicillin resistant colony containing plasmid pMeJBint wasisolated. This plasmid was digested with ClaI and treated with bacterialalkaline phosphatase in order to reduce self ligation and is designatedfragment 3. Plasmid pMe2 was digested with ClaI and the smaller fragment(fragment 4) was isolated. This fragment contains the Mucor miehei acidprotease 3′ codons and about 1800 bp of 3′ flanking sequences. Fragments3 and 4 were joined by ligation and used to transform E. coli 294. Oneampicillin resistant colony containing plasmid pMeJB1-7 was isolated.This vector was used to transform Aspergillus nidulans.

[0142] D. Expression and Secretion of Mucor miehei Carboxyl Protease byAspergillus Nidulans

[0143] Southern blot analysis of six transformants indicated thepresence of the entire Mucor miehei acid protease gene in theAspergillus nidulans genome (results not shown). In addition, each ofthe transformants were analyzed by Western blots (results not shown) andfor acid protease activity. The results of the protease assay are shownin Table VI. TABLE VI Protease Transformant Activity (mg/ml) 1 0.003 20.007 3 0.003 4 0.005 5 0.005 6 0.012

[0144] These experiments demonstrate expression and secretion of aprotein that reacts with specific antibody to Mucor miehei carboxylprotease and which has milk clotting activity. The protein has anapparent molecular weight by electrophoretic analysis that is slightlygreater than that of the authentic (Mucor miehei derived) protein. Thismay indicate that Aspergillus nidulans glycosylates this glycoprotein toa different extent than Mucor miehei. Because the Aspergillus nidulansderived Mucor meihei acid protease appears to have the same specificactivity as the authentic material it appears that it has been processed(by the cell or autocatalytically) to the mature form. The unprocessedforms of other acid proteases such as chymosin and pepsin are zymogenswhich require processing (autocatalytic) before activity is obtained.

[0145] The varying levels of expression in the various transformants mayreflect the position or copy number of the protease gene in theAspergillus nidulans genome. However, the expression and secretion ofbiologically active carboxyl protease indicates the A. nidulansrecognizes the promoter, signal and terminator signals of Mucor mieheicarboxyl protease.

EXAMPLE 7

[0146] Expression and Secretion of Chymosin Encoded by pGRG1-4 from A.Awamori and Trichoderma reesei pyrG Auxotrophic Mutants

[0147] The plasmids pGRG1 through pGRG4 (pGRG1-4) were also used totransform orotidine-5′-phosphate decarboxylase (OMPCase) deficientmutants of A. awamori and Trichoderma reesei. The pyr4 gene from N.crassa encoded by the pGRG1-4 plasmid complements these OMPCase mutantsin the absence of uridine to permit the isolation of successfultransformants. The thus transformed mutants of A. awamori and T. reeseisecreted detectable amounts of bichemically active chymosin into theculture medium.

[0148] A. Production of pyrG Auxotrophs

[0149] The method used to obtain pyrG-auxotrophic mutants of A. awamoriand T. reesei involved selection on the pyrimidine analog5-fluoro-orotic acid (FOA) (56). The mechanism by which FOA killswild-type cells is unknown. However, in view of the resistance ofOMPCase-deficient mutants to FOA, it is likely that the toxicity occursthrough conversion of FOA to 5-fluoro-UMP. Whether cell death is causedby a flouoridated ribonucleotide or deoxyribonucleotide is uncertain.

[0150] The following methods describe the isolation of OMPCase-deficient(FOA-resistant) mutants of T. reesei and A. awamori:

[0151] 1. Trichoderma reesei

[0152] A fresh spore suspension of T. reesei strain P37 (NRRL 15709) waswashed three times in sterile distilled water containing 0.01% Tween-80.Fifteen milliliters of this spore suspension (1×10⁷ spores per ml) wereplaced in a sterile petri dish (100×20 mm) with a sterile magneticstirring bar. The lid was removed and the spores were exposed toultraviolet (UV) light at 254 nm (7000 uW per cm2), in the dark at adistance of 25 cm from the UV light source. The spores were stirredconstantly. UV exposure continued for three minutes (sufficient to give70% killed spores). The irradiated spore suspension was collected in a50 ml centrifuge tube and stored in the dark for one hour to preventphotoreactivation. Spores were pelleted by centrifugation and the pelletwas resuspended in 200 ul of sterile water containing 0.01% Tween-80.

[0153] The suspension was diluted and plated onto YNB agar medium (0.7%yeast nitrogen base without amino acids, 2% glucose, 10 mM uridine, 2%agar) (56) containing 0.15% FOA (SCM Specialty Chemicals, Gainsville,Florida). After 4 days incubation at 30° C., 75 colonies were picked tofresh YNB agar that contained FOA. Sixty-two of the 75 colonies grew andwere toothpicked to minimal agar (6 g/l NaNO₃, 0.52 g/l KCl, 1.52 g/lKH₂PO₄1 ml/l trace elements solution, 1% glucose, 0.1% MgSO₄, 20 g/lagar) and minimal agar plus 1 mg/ml uridine to determine uridinerequirements. All of the 62 isolates grew on minimal agar with uridine,but 9 isolates failed to grow on minimal agar alone. These 9 strainswere repicked to minimal agar and minimal agar with uridine. Two of thestrains grew only on minimal agar supplemented with uridine. One ofthese, designated T. reesei pyrG29, grew well on minimal medium withuridine with no background growth on minimal medium alone.

[0154] 2. Aspergillus awamori

[0155] (i) Production of A. awamori strain UVK 143f- a Hyperproducer ofGlucoamylase

[0156] Spores of A. awamori strain NRRL 3112 were obtained after 5-7days growth on Potato Dextrose Agar (PDA, Difco Co.) at 30° C. Sporeswere harvested by washing the surface of the plate with sterile 0.1%Tween-80 in distilled H₂O and gently scraping the spores free. Sporeswere washed by centrifugation and resuspended in the same buffer to givea final concentration of between 1×10⁷ to 2×10⁸ spores/ml. Preparationswere stored at 4° C.

[0157] Two ml of spores was added to a sterile petri plate. The top ofthe dish was removed and spores were exposed to an ultraviolet (UV) lamp(15 watt, germicidal). Conditions of time exposure and distance from thelamp were adjusted such that 90 to 99.9% of the spores were killed.Surviving spores were plated onto PDA medium and grown to form discreteindependent colonies.

[0158] Spores from individual mutagenized colonies were inoculated into50 ml of screening media consisting of 5% corn meal, 0.5% yeast extract,2% corn steep liquor, adjusted to pH 4.5 prior to sterilization in 250ml flasks. However, any number of media containing corn or corn starchas the carbon source would be expected to give similar results. Cultureswere grown for 4-5 days at 30-35° C. with constant shaking. Samples wereremoved either daily or at the end of the run for assays.

[0159] Estimates of glucoamylase activity were made by measuring therelease of a color producing group (para-nitro-phenol) from a colorlesssubstrate (para-nitro-phenyl-alpha-glucoside, PNPAG).

[0160] The following protocol was utilized:

[0161] Substrate—180 mg PNPAG was dissolved in 250 ml of 0.1M NaAcetatebuffer, pH 4.7. Store at 4° C.

[0162] Assay—1 ml of substrate was equilibrated at 40° C. in a waterbath. 0.2 ml of sample (or diluted sample) was added and incubated for30 minutes at 40° C. 9 ml of 0.1M Na₂C0₃ was added with the mixturebeing kept at room temperature for 15 minutes for color development. Themixture was filtered through Wattman 42 filter paper and the absorbanceat 420 nm was read in a suitable spectrophotometer. All mutant PNPAGlevels were compared to the standard amount produced by the parentstrain and were reported as percent of PNPAG hyrolysis of the parent.

[0163] One glucoamylase-hyperproducing strain designated UVK 143f wasselected for auxotrophic mutagenesis.

[0164] (ii) Auxotrophic Mutagenesis

[0165] Preparation of spores from A. awamori strain UVK143f, UVmutagenesis, and mutant analysis were the same as for T. reesei with thefollowing modifications:

[0166] a. 2.5 minutes was required to give 70% killing with UV light.

[0167] b. Minimal medium was used instead of YNB agar.

[0168] c. The FOA concentration was 0.1%.

[0169] Fifteen pyrG mutants were found. Three of these isolates,designated pyr4-5, pyr4-7, and pyr4-8 were selected for transformationexperiments.

[0170] B. Transformation of A. awamori and T. reesei pyr Auxotrophs

[0171]A. awamori and T. reesei auxotrophs were transformed by amodification of the procedure previously described for A. nidulans.Approximately 1×10⁸ spores were inoculated into yeast extract glucose(YEG) medium which is 2% glucose, 0.5% yeast extract supplemented with 1mg/ml uridine. The cultures were incubated on a 37° C. shaker (200 rpm)for 12 to 15 hours [T. reesei was incubated at 30° C.]. Germlings wereharvested by centrifugation, washed once with sterile YEG medium, thenincubated at 30° C. in 50% YEG medium containing 0.6 M KCl, 0.5%Novozyme 234 (Novo Industries, Denmark), 0.5% MgSO₄.7H₂O, 0.05% bovineserum albumin in a sterile 200 ml plastic bottle (Corning Corp.,Corning, N.Y.). After 30 minutes of shaking at 150 rpm, the protoplastsuspension was vigorously pipetted up and down five times with a 10 mlpipette to disperse the clumps. The protoplast suspension was furtherincubated as above for one hour then filtered through sterile miracloth(Calbiochem-Behring Corp., LaJolla, Calif.) that was wet with 0.6 M KCl.The filtered protoplasts were centrifuged, washed, and transformed witheach of the plasmids pGRG1-4 as described previously.

[0172] The following modifications were made for A. awamoritransformation:

[0173] 1. 0.7 M KCl was used instead of 0.6 M KCl.

[0174] 2. 1.2 M sorbitol was used instead of 0.6 M KCl to osmoticallystabilize the transformation and regeneration medium.

[0175] C. Analysis of A. awamori and T. reesei Transformants

[0176] Both A. awamori and T. reesei transformants secreted chymosinpolypeptides into the culture medium. This was determined by analyzingculture filtrates (results not shown) for both enzymatically activechymosin (milk clotting assay) and chymosin polypeptides that reactedwith specific chymosin antibodies (enzyme immunoassays and Westernimmunoblotting techniques).

EXAMPLE 8

[0177] Expression and Secretion of Heterologous Polypeptides from argBAuxotrophic Mutants of Aspergillus Species

[0178] The expression and secretion of heterologous polypeptides fromargB auxotrophs of Aspergillus species has also been achieved.

[0179] This example describes the complementary transformation of A.nidulans and A. awamori argB auxotrophs with vectors containing the argBgene from A. nidulans and DNA sequences encoding the heterologouspolypeptides of plasmids pGRG1-4. The argB gene encodes ornithinetranscarbamylase (OTC).

[0180] The A. nidulans argB auxotroph containing the genetic markersbiA1, argB2, metG1 used herein was obtained from Dr. P. Weglenski,Department of Genetics, Warsaw University, Al. Ujazdowskie 4,00-478Warsaw, Poland. The A. awamori argB mutant was derived as follows.

[0181] A. Isolation of Aspergillus awamori argB Auxotrophic Mutants

[0182] A fresh suspension of A. awamori strain UVK 143 k spores wasprepared and UV mutagenesis was performed as described above except thatthe exposure time was sufficient to kill 95% of the spores. The sporeswere then centrifuged, washed with sterile water, and resuspended in 25ml of sterile minimal medium. These suspensions were incubated in a 37°C. shaker with vigorous aeration. Under these conditions, wild-typespores will germinate and grow into vegetative mycelia, but auxotrophicmutants will not. The culture was aseptically filtered through sterilemiracloth every six to eight hours for three days. This step removesmost of the wild-type mycelia while the ungerminated auxotrophs passthrough the miracloth filter (i.e., filtration enrichment). At eachfiltration step the filtered spores were centrifuged and resuspended infresh minimal medium. After three days of enrichment the spores werediluted and plated on minimal agar supplemented with 50 mM citrulline.The plates were incubated at 37° C. for two to three days. Individualcolonies were toothpicked from these plates to two screening plates—oneplate that contained minimal agar plus 10 mM orhithine and one platethat contained minimal agar plus 50 mM citrulline. The rationale forpicking colonies to these screening plates is as follows. OTC (the argBgene product) catalyzes the conversion of ornithine to citrulline in thearginine biosynthetic pathway. Thus argB mutants (deficient in OTC) willgrow on minimal medium plus citrulline but not on minimal medium withornithine. Screening of approximately 4000 colonies by this methodyielded 15 possible argB mutants. One of these strains, designated A.awamori argB3, gave no background growth on minimal medium and grew verywell on minimal medium supplemented with either arginine or citrulline.Assays to determine the level of OTC activity (57) indicated that theargB3 mutant produced at least 30-fold less OTC activity than wild-type.On the basis of these data the A. awamori argB3 strain was selected fortransformation experiments.

[0183] B. Construction of argB-based Prochymosin Expression Vectors forTransformation of Aspergillus Species

[0184] In this construction (see FIG. 15) the first step was to combinethe transformation enhancing sequence ANS-1 and the selectable argB geneon the same plasmid. In order to achieve this, plasmid pBB116 (59),which contains the argB gene from A. nidulans, was digested with PstIand BamHI and the indicated fragment A, which contains the argBstructural gene, was isolated. Plasmid pDJB2 (59) was digested withEcoRl and PstI, and the indicated fragment B, which contains the ANS-1sequence, was isolated. In a three part ligation fragments A and B werejoined together with fragment C, which contains the large EcoRl-BamHIfragment of plasmid vector pUC18 (33) to give plasmid pARG-DJB.

[0185] In the second step of this construction a synthetic DNApolylinker containing ClaI sites was inserted into pARG-DJB in order toallow the insertion of ClaI fragments which contain various prochymosinexpression units. Plasmid pARG-DJB was digested with BamHI and thendephoshporylated with bacterial alkaline phosphatase. The indicatedsynthetic DNA polylinker was phosphorylated with T4 polynucleotidekinase, and then ligated to the cleaved pARG-DJB to give pCJ16L. Becausethis plasmid was found to be resistant to digestion with ClaI, it wasfirst used to transform the E. coli dam-mutant strain GM48 in order toprevent methylation of the ClaI sites. Upon isolation of the plasmidfrom GM48 transformants, it was successfully cleaved with ClaI anddephosphorylated with bacterial alkaline phosphatase.

[0186] In the final step of this construciton of ClaI-cleaved pCJ16Lvector was joined to each of the ClaI prochymosin expression fragmentsfrom plasmids pGRG1 through pGRG4. The resulting four plasmids,pCJ::GRG1 through pCJ::GRG4, were used to transform the argB mutants ofA. nidulans and A. awamori to prototrophy. Resulting transformants wereanalyzed for expression of prochymosin polypeptides.

[0187] C. Analysis of A. nidulans and A. awamori Transformants

[0188] Secreted chymosin polypeptides from A. awamori and A. nidulanstransformed with pCJ::GRG1 through pCJ::GRG4 were detected by the milkclotting assay and by enzyme immunoassays and Western immunoblottingtechniques. In each case (results not shown) the transformed fungisecreted biochemically active chymosin into the culture medium.

EXAMPLE 9

[0189] Expression and Secretion of Humicola grisea glucoamylase from A.nidulans

[0190] The glucoamylase gene from the fungus Humicola grisea wasisolated and cloned. This gene was thereafter ligated into the argBexpression plasmid pCJ16L. The resulting vectors, pCJ:RSH1 and pCJ:RSH2were used to transform argB deficient A. nidulans (Example 8) whichresulted in the expression and secretion of Humicola griseaglucoamylase.

[0191] A. Isolation and Cloning of Humicola grisea Glucoamylase Gene

[0192] 1. Purification of Humicola grisea Glucoamylase

[0193] Authentic H. grisea (var. thermoidea NRRL 15219) glucoamylase wasobtained from A. E. Staley Company (lot no. 1500-149-8A). The enzyme waspurified to homogeneity through chromatography on a 4.6 mm×250 mmSynchrompak C4 reversed phase column (SynChrom, Inc., Linden, Ind.). Thecolumn was initially equilibrated with 0.05% triethylamine and 0.05%trifluoroacetic acid (solvent A) at 0.5 ml/min. After injection of theglycomaylase sample (40 μg) the column was washed for 2 minutes withsolvent A, and then eluted with a gradient of 5% solvent B per minute(solvent B is 0.05% triethylamine, 0.05% trifluoroacetic acid inacetonitrile) to 40% solvent B. The slope of the gradient was thenchanged to 0.5% solvent B per minute, and the glucoamylase was eluted atapproximately 55% solvent B. At this point the glucoamylase was judgedto be homogenous by sodium dodecylsulfate polyacrylamide gelelectrophoresis.

[0194] 2. Amino Acid Sequence of H. grisea Glucoamylase

[0195] The amino terminal sequence of purified H. grisea glucoamylasewas obtained as described previously (60). The sequence read as follows:

[0196] AAVDTFINTEKPSAXNSL

[0197] These and other lettered peptide sequences presented herein referto amino acid sequences wherein each letter corresponds to the followingamino acids: Amino acid or 3-letter 1-letter residue thereof symbolsymbol Alanine Ala A Glutamate Glu E Glutamine Gln Q Aspartate Asp DAsparagine Asn N Leucine Leu L Glycine Gly G Lysine Lys K Serine Ser SValine Val V Arginine Arg R Threonine Thr T Proline Pro P Isoleucine IleI Methionine Met M Phenylalanine Phe F Tyrosine Tyr Y Cysteine Cys CTryptophan Trp W Histidine His H

[0198] In order to obtain peptide fragments for additional amino acidsequencing, purified glucoamylase (1 mg/ml) was digested in 2% aceticacid for 2 hours at 108° C. The material was injected directly onto aSynchrompak C4 column (4.8 mm×100 mm) equilibrated as described above.After washing for 2 minutes with 100% solvent A (see above), thepeptides were eluted with a linear gradient of solvent C (1% perminute). Solvent C was composed of 0.05% triethylamine, 0.05%trifluoroacetic acid in propanol. At this point three peptides wereselected for futher analysis. One peptide (GA3) was sequenced directly.A mixture of two other peptides (GA1 and GA2) was subjected to furtherpurification on a 4.8 mm×250 mm Synchrompak C4 column as follows. Themixture of GA1 and GA2 was diluted with three volumes of solvent A andinjected onto the column. After washing for 2 minutes, the peptides wereeluted with a linear gradient of solvent D (0.5% per minute). Solvent Dwas 0.05% triethylamine, 0.05% trifluoroacetic acid in 35% propanol:65%acetonitrile. Separated GA1 and GA2 were then purified again using thesame protocol and the amino acid sequences were determined as describedabove. The sequences of peptides GA1, GA2 and GA3 are as follows: GA1PLWSITVPIKATGXAVQYKYIKVXQL GA2 AAVRPLINPEKPIAWNXLKANIGPN GA3INTEKPIAWNKLLANIGPNGKAAPGAAAGVVIASPSRTD

[0199] 3. Synthetic Oligonucleotide Probes

[0200] The genomic DNA encoding the H. grisea glucoamylase gene wascloned as follows. A synthetic mixture of 48 oligonucleotides was usedas a hybridization probe to detect the glucoamylase gene. Theoligonucleotides were 17 bases in length (17 mer) and corresponded to asequence of six amino acids (underlined in the GA1 peptide, supra) fromH. grisea glucoamylase:    Gln Tyr Lys Tyr Ile Lys 5′ CAA TAT AAA TATATT AA 3′      G   C   G   C   C                      A

[0201] The mixture of 48 oligonucleotides was synthesized in six pools,each containing eight different synthetic 17 mers. pool 1:5′ CAATATAAATATATTAA 3′       G  C  G pool 2: 5′ CAATATAAATACATTAA 3′     G  C  G pool 3: 5′ CAATATAAATATATCAA 3′      G  C  G pool 4:5′ CAATATAAATACATCAA 3′      G  C  G pool 5: 5′ CAATATAAATATATAAA 3      G  C  G pool 6: 5′ CAATATAAATACATAAA 3′      G  C  G

[0202] The oligonucleotides were synthesized on a Biosearch automatedDNA synthesizer (Biosearch, San Rafael, Calif.) using reagents andprotocols specified by the manufacturer.

[0203] 4. Selection of Correct Oligonucleotide Probe

[0204] Genomic DNA from H. grisea was analyzed for the presence ofglucoamylase sequences by the method of Southern (30). Briefly, H.grisea DNA was digested with BamHI restriction endonuclease. Sixaliquots of this digested DNA (one for each probe pool) werefractionated according to size by electrophoresis on a 1% agarose gel.After blotting the DNA to nitrocellulose, as previously described, theDNA was fixed to the nitrocellulose filter at 80° C. in a vacuum oven.The filter was cut into six strips, corresponding to the six aliquots ofBamHI digested DNA, and each strip was hybridized for 18 hours at lowstringency (2, 40) with one of the pools of synthetic oligonucleotideprobes. (The probes were radiolabeled with gamma-[32P]ATP using T4polynucleotide kinase as previously described.) After hybridization, thefilters were washed 15 minutes in 2×SSC, 0.1% SDS at 37° C., and twicein 2×SSC at the same temperature. Filters were air dried, wrapped inSaran-Wrap, and applied to Kodak XOmat-AR X-ray film to obtain anautoradiographic image. After developing the autoradiogram, a faint bandof hybridization corresponding to a 3.7 Kb BamHI fragment was visiblefrom the strip that was hybridized with pool 3.

[0205] In order to improve the hybridization signal, pool 3 wasre-synthesized as eight individual oligonucleotides. The Southernhybridization experiments were repeated using each of the eightoligonucleotides as probes. Only one of these 17 mer probes was found tohybridize to the 3.7 Kb BamHI fragment of H. grisea genomic DNA. Thesequence of the oligonucleotide was 5′CAGTACAAGTATATCAA 3′. This 17 merwas used as the hybridization probe for the cloning of the H. griseaglucoamylase gene.

[0206] 5. Cloning of Glucoamylase Gene Sequences

[0207] Genomic DNA from H. grisea was digested with BamHI andsize-fractionated by polyacrylamide gel electrophoresis according tostandard methods (30). DNA fragments 2 to 4 Kb in size were excised andeluted from the gel. This DNA fraction was used to generate a library ofclones in the E. coli cloning vector pBR322 (ATCC 37017). The cloningvector was digested with BamHI and dephosphorylated with bacterialalkaline phosphatase. The phosphatase was removed by extraction withphenol-chloroform (1:1 v/v). The BamHI cleaved size-selected H. griseaDNA was ligated to the BamHI cleaved and dephosphorylated pBR322. Thethus ligated DNA was used to transform competent E. coli 294 cells (ATCC31446) prepared by the method of Morrison (41). Transformants wereselected on LB agar plates (30) which contained carbenecillin at aconcentration of 50 μg/ml. Transformants which harbored glucoamylasegene sequences were identified by colony hybridization methods (30)using the specific 17 mer (described above) as a probe. Hybridizingcolonies were purified, and plasmids were isolated from each by thealkaline-SDS miniscreen procedure (30). The plasmids selected in thismanner all contained a 3.7 Kb BamHI fragment which hybridized to theglucoamylase-specific 17mer probe. One such plasmid, designated pRSH1,was selected for further analysis.

[0208] A 600 bp Sau3A fragment from pRSH1 was subcloned intobacteriophage M13 mp18 (33) and partially sequenced by the dideoxy chaintermination method (43) to confirm that the cloned DNA encoded theglucoamylase gene. A restriction endonuclease cleavage map of the 3.7 KbBamHI fragment contained in pRSH1 is shown in FIG. 16. It was generatedfollowing single and double restriction digests followed by orientationof the DNA fragments with respect to known restriction sites in pBR322(44). On the basis of the DNA sequencing data we obtained and therestriction map, we determined that there was a high probability thatthe entire coding sequence of the glucoamylase gene was contained withinthe 3.7 Kb BamHI fragment in pRSH1.

[0209] B. Construction of argB Vector Containing Humicola griseaGlucoamylase Gene

[0210] The 3.7 Kb BamHI fragment from pRSH1 was cloned (in bothorientations) into pCJ16L which contains a selectable argB gene from A.nidulans (FIG. 17). The resulting vectors, PCJ:RSH1 and pCJ:RSH2, wereused to transform argB-deficient A. nidulans.

[0211] C. Expression and Secretion of H. grisea glucoamylase

[0212] Prototrophic transformants were purified and innoculated intominimal medium with starch as the sole carbon source (this medium is thesame as that described for the production of chymosin except that the pHwas adjusted to 5.0). Culture filtrates were assayed for H. griseaglucoamylase activity. FIG. 18 shows the extracellular production of H.grisea glucoamylase by A. nidulans transformed with pCJ:RSH1. Thenegative control was non-transformed argB deficient A. nidulans.

[0213] Although the foregoing refers to particular preferredembodiments, it will be understood that the present invention is not solimited. It will occur to those ordinarily skilled in the art thatvarious modifications may be made to the disclosed embodiments and thatsuch modifications are intended to be within the scope of the presentinvention.

[0214] The references grouped in the following bibliography andrespectively cited parenthetically by number in the foregoing text, arehereby incorporated by reference.

Bibliography

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1 28 1 24 DNA Artificial Sequence phospharylated synthetic linkersequence 1 gatccatcga tctcgagatc gatg 24 2 1900 DNA Rhizomucor mieheiCDS (408)..(1700) 2 aagcttccat ttcagaaaag aaagctctcg gagttgtcattgaggcattt ttacagcctt 60 tttccgctct cactctgtat ttctatgaga aagagaaagataggaatgtc atgcataaga 120 tgctacaaga tcatgccaac ggtagtacac acaattcctgccttttatga cctttttatg 180 ctaagtcttt ttggaaattt atcccgcggt cctaggttgtacccagtcgt tgcatccacc 240 tttcctggta aaaacataca attatggact aaatggtttcttaaatgtca acactctaga 300 cggaagggca caaagatctt tagtgcctgg gccaactgtaggtagatctt tttttcatat 360 aaaaccagac gagtgtgaag gttgcgagac tccatcagattccgacc atg ctc ttc 416 Met Leu Phe 1 tct cag att act tct gcg atc ctttta aca gcg gct tcc ttg tcg ctt 464 Ser Gln Ile Thr Ser Ala Ile Leu LeuThr Ala Ala Ser Leu Ser Leu 5 10 15 acc act gct cgc ccg gta tcc aag caatcc gag tcc aag gac aag ctt 512 Thr Thr Ala Arg Pro Val Ser Lys Gln SerGlu Ser Lys Asp Lys Leu 20 25 30 35 ctg gcg ctt cct ctc acc tcg gtg tcccgc aag ttc tct caa acc aag 560 Leu Ala Leu Pro Leu Thr Ser Val Ser ArgLys Phe Ser Gln Thr Lys 40 45 50 ttc ggt cag caa caa ctt gct gag aag ctagca ggt ctc aag ccc ttc 608 Phe Gly Gln Gln Gln Leu Ala Glu Lys Leu AlaGly Leu Lys Pro Phe 55 60 65 tct gaa gct gcc gca gac ggc tcc gtc gat acgccc ggc tat tac gac 656 Ser Glu Ala Ala Ala Asp Gly Ser Val Asp Thr ProGly Tyr Tyr Asp 70 75 80 ttt gat ctg gag gag tat gct att ccg gtc tcc attggt act cct ggt 704 Phe Asp Leu Glu Glu Tyr Ala Ile Pro Val Ser Ile GlyThr Pro Gly 85 90 95 caa gac ttt ttg ctc ttg ttc gac act ggc agc tcc gatact tgg gtt 752 Gln Asp Phe Leu Leu Leu Phe Asp Thr Gly Ser Ser Asp ThrTrp Val 100 105 110 115 cca cac aag ggt tgc acc aag tct gaa ggt tgt gttggc agc cga ttc 800 Pro His Lys Gly Cys Thr Lys Ser Glu Gly Cys Val GlySer Arg Phe 120 125 130 ttt gat cca tcg gct tcc tcc act ttt aaa gca actaac tac aac cta 848 Phe Asp Pro Ser Ala Ser Ser Thr Phe Lys Ala Thr AsnTyr Asn Leu 135 140 145 aac atc acc tac ggt act ggc ggc gca aac ggt ctttac ttt gaa gac 896 Asn Ile Thr Tyr Gly Thr Gly Gly Ala Asn Gly Leu TyrPhe Glu Asp 150 155 160 agc atc gct atc ggc gac atc acc gtg acc aag caaatt ctg gct tac 944 Ser Ile Ala Ile Gly Asp Ile Thr Val Thr Lys Gln IleLeu Ala Tyr 165 170 175 gtc gat aat gtt cgc ggc cca act gct gag cag tctcct aac gct gac 992 Val Asp Asn Val Arg Gly Pro Thr Ala Glu Gln Ser ProAsn Ala Asp 180 185 190 195 att ttc ctt gat ggt ctc ttt ggt gca gcc taccca gac aac acg gcc 1040 Ile Phe Leu Asp Gly Leu Phe Gly Ala Ala Tyr ProAsp Asn Thr Ala 200 205 210 atg gaa gca gag tat gga tcg act tat aac actgtt cac gtc aac ctc 1088 Met Glu Ala Glu Tyr Gly Ser Thr Tyr Asn Thr ValHis Val Asn Leu 215 220 225 tac aag caa ggc ttg atc tct tct cct ctt ttctcg gtc tac atg aac 1136 Tyr Lys Gln Gly Leu Ile Ser Ser Pro Leu Phe SerVal Tyr Met Asn 230 235 240 act aac agc ggc act gga gag gtc gtc ttt ggtgga gtc aac aac acg 1184 Thr Asn Ser Gly Thr Gly Glu Val Val Phe Gly GlyVal Asn Asn Thr 245 250 255 ctt ctc ggc ggc gac att gcc tac acg gac gttatg agt cgt tat ggt 1232 Leu Leu Gly Gly Asp Ile Ala Tyr Thr Asp Val MetSer Arg Tyr Gly 260 265 270 275 ggt tat tac ttc tgg gac gca ccc gtc acaggt atc acc gtc gat gga 1280 Gly Tyr Tyr Phe Trp Asp Ala Pro Val Thr GlyIle Thr Val Asp Gly 280 285 290 tct gct gct gtc agg ttc tcg aga ccc caagca ttc acc atc gat act 1328 Ser Ala Ala Val Arg Phe Ser Arg Pro Gln AlaPhe Thr Ile Asp Thr 295 300 305 ggc acc aac ttt ttc att atg ccc tca agcgcc gct tct aag att gtc 1376 Gly Thr Asn Phe Phe Ile Met Pro Ser Ser AlaAla Ser Lys Ile Val 310 315 320 aaa gca gct ctc cct gat gcc act gaa acccag cag ggc tgg gtt gtt 1424 Lys Ala Ala Leu Pro Asp Ala Thr Glu Thr GlnGln Gly Trp Val Val 325 330 335 cct tgc gct agc tac cag aac tcc aag tcgact atc agc atc gtc atg 1472 Pro Cys Ala Ser Tyr Gln Asn Ser Lys Ser ThrIle Ser Ile Val Met 340 345 350 355 caa aag tcc ggc tca agc agt gac actatt gag atc tcg gtt cct gtc 1520 Gln Lys Ser Gly Ser Ser Ser Asp Thr IleGlu Ile Ser Val Pro Val 360 365 370 agc aaa atg ctt ctt cca gtc gac caatcg aac gag act tgc atg ttt 1568 Ser Lys Met Leu Leu Pro Val Asp Gln SerAsn Glu Thr Cys Met Phe 375 380 385 atc att ctt ccc gac ggt ggt aac cagtac att gtt ggc aac ttg ttc 1616 Ile Ile Leu Pro Asp Gly Gly Asn Gln TyrIle Val Gly Asn Leu Phe 390 395 400 ctg cgc ttc ttt gtc aat gtt tac gacttt ggc aac aac cgt atc ggc 1664 Leu Arg Phe Phe Val Asn Val Tyr Asp PheGly Asn Asn Arg Ile Gly 405 410 415 ttt gca cct ttg gcc tcg gct tat gaaaac gag taa aggggcacca 1710 Phe Ala Pro Leu Ala Ser Ala Tyr Glu Asn Glu420 425 430 attcttcttt agctgctcag ataactttgt aactctctga tatactctttataaccttta 1770 tttctcactt tttaactgta ttccaataca ttatttgaac ttactaaatattacgcttat 1830 tcttgtttgg gtgagttgta gagtaaaaaa aatgcttaga agcggaattgtattttgcaa 1890 ggaatgtaca 1900 3 430 PRT Rhizomucor miehei 3 Met LeuPhe Ser Gln Ile Thr Ser Ala Ile Leu Leu Thr Ala Ala Ser 1 5 10 15 LeuSer Leu Thr Thr Ala Arg Pro Val Ser Lys Gln Ser Glu Ser Lys 20 25 30 AspLys Leu Leu Ala Leu Pro Leu Thr Ser Val Ser Arg Lys Phe Ser 35 40 45 GlnThr Lys Phe Gly Gln Gln Gln Leu Ala Glu Lys Leu Ala Gly Leu 50 55 60 LysPro Phe Ser Glu Ala Ala Ala Asp Gly Ser Val Asp Thr Pro Gly 65 70 75 80Tyr Tyr Asp Phe Asp Leu Glu Glu Tyr Ala Ile Pro Val Ser Ile Gly 85 90 95Thr Pro Gly Gln Asp Phe Leu Leu Leu Phe Asp Thr Gly Ser Ser Asp 100 105110 Thr Trp Val Pro His Lys Gly Cys Thr Lys Ser Glu Gly Cys Val Gly 115120 125 Ser Arg Phe Phe Asp Pro Ser Ala Ser Ser Thr Phe Lys Ala Thr Asn130 135 140 Tyr Asn Leu Asn Ile Thr Tyr Gly Thr Gly Gly Ala Asn Gly LeuTyr 145 150 155 160 Phe Glu Asp Ser Ile Ala Ile Gly Asp Ile Thr Val ThrLys Gln Ile 165 170 175 Leu Ala Tyr Val Asp Asn Val Arg Gly Pro Thr AlaGlu Gln Ser Pro 180 185 190 Asn Ala Asp Ile Phe Leu Asp Gly Leu Phe GlyAla Ala Tyr Pro Asp 195 200 205 Asn Thr Ala Met Glu Ala Glu Tyr Gly SerThr Tyr Asn Thr Val His 210 215 220 Val Asn Leu Tyr Lys Gln Gly Leu IleSer Ser Pro Leu Phe Ser Val 225 230 235 240 Tyr Met Asn Thr Asn Ser GlyThr Gly Glu Val Val Phe Gly Gly Val 245 250 255 Asn Asn Thr Leu Leu GlyGly Asp Ile Ala Tyr Thr Asp Val Met Ser 260 265 270 Arg Tyr Gly Gly TyrTyr Phe Trp Asp Ala Pro Val Thr Gly Ile Thr 275 280 285 Val Asp Gly SerAla Ala Val Arg Phe Ser Arg Pro Gln Ala Phe Thr 290 295 300 Ile Asp ThrGly Thr Asn Phe Phe Ile Met Pro Ser Ser Ala Ala Ser 305 310 315 320 LysIle Val Lys Ala Ala Leu Pro Asp Ala Thr Glu Thr Gln Gln Gly 325 330 335Trp Val Val Pro Cys Ala Ser Tyr Gln Asn Ser Lys Ser Thr Ile Ser 340 345350 Ile Val Met Gln Lys Ser Gly Ser Ser Ser Asp Thr Ile Glu Ile Ser 355360 365 Val Pro Val Ser Lys Met Leu Leu Pro Val Asp Gln Ser Asn Glu Thr370 375 380 Cys Met Phe Ile Ile Leu Pro Asp Gly Gly Asn Gln Tyr Ile ValGly 385 390 395 400 Asn Leu Phe Leu Arg Phe Phe Val Asn Val Tyr Asp PheGly Asn Asn 405 410 415 Arg Ile Gly Phe Ala Pro Leu Ala Ser Ala Tyr GluAsn Glu 420 425 430 4 1864 DNA Emericella nidulans 4 cagctgacttgataattaca ttactataaa aaactcttac taagttacta gataagaact 60 ttatttatattagttactta tttatagtaa ctttagtact acttataaaa aagctagata 120 gagatttatagttctatact aattactatg ccttaaatat tattattaaa aaagattatt 180 atcccctttctctaattaat aaaactctag aatttatctc taaaattaaa taatttataa 240 aacttaatattattactatc ttttataaaa tctagattta ttttaaagat aaatagaaga 300 tagctttcaggatatatttt aggttatata aatagttaat tacttttttt aagctagtaa 360 atattctaagtatattctaa tattatatta actagatctt acataacttt ctagataaat 420 ttgcctttatatacttagat aatatattaa tttttactaa tagcttatta aaaaagtatt 480 ataattatatataattaatt ttctagaaac taggcaaagc aggactatag cttaatatta 540 ataaatataattttaaaatt aactaagtct ttaagattta ttattaatat aaagaagggt 600 atataaatagatcctaagaa aataagctat ccttaaataa taaatcctta tattagttaa 660 aaaaaatatacttcttttta agatttataa atttttatta atttatataa aacttcttag 720 acctgatatacctacttact attctaatat aaaaaattat taaattttta tagattaaga 780 agtatacctagtttaaatag ctaaattaaa tatttataat aacttctatt tttatataat 840 ttaatcctaattaagaaata atttttaaag ctcttatatt aaactagact actagaaaaa 900 tattattataatataataat aataaagtac tatattttta tatttattat ttaagaactc 960 tccctgcaaaatataattat aaaatttata ataaaaatta ttagtaatta ttaatagctt 1020 aaaagtttagaaattagagc ttataagttt aaaaaatttt attattatta taaattataa 1080 aaacctactatactttacta taattagata ttttaataaa tagtaaatat attagacaga 1140 tatacttaattattttaatt ttattttaaa tattaactag gtaagctagc agctttgcct 1200 agatatattattatactata aataagatat actagccagg gtagacaata attaacttaa 1260 aatatataaaaaatagctac taaaactgaa taatattaaa gaatagtatt tatctaagta 1320 taacttaaatcttagtaata ctaaagaatt taattaatta attaaaatat ataaatatag 1380 ttgaccttgaaactgttact aaactttatt ataatattat taattataat aactaagcta 1440 agaataaattacttaagaaa ctataaaaaa tagtctagga gaataataaa gctctataaa 1500 aaattattaagattattaag aagaatagat agagcttcct aagagattac aaatatatat 1560 cttattttctaaatatttaa tattatctag agataatttt ttttattctt aattttaggt 1620 cctgggaagtaaacctttaa aaacctaaat tatatagatc tggattaact agctaccctg 1680 ggcaaaacagcctatatatt atatatatta attcttaata atctagtagt atatcttttt 1740 tacctattatagatcaagag attaaaacta gctagggcta atatataata aaatttgttt 1800 ttacttagttacttattagt ttgtcaatcc gcaccgcaac ccgcagcggg tcaccacact 1860 gcag 1864 528 DNA Aspergillus niger 5 atgtcgttcc gatctctact cgccctga 28 6 10 PRTAspergillus niger 6 Met Ser Phe Arg Ser Leu Leu Ala Leu Ser 1 5 10 7 7DNA Unknown eukaryotic promoter sequence 7 tataaat 7 8 4 DNA Unknowneukaryotic promoter sequence 8 caat 4 9 28 DNA Artificial Sequencesynthetic oligonucleotide 9 gctcggggtt ggcagctgag atcaccag 28 10 28 DNAArtificial Sequence synthetic oligonucleotide 10 actcccccac cgcaatgaggtgtctcgt 28 11 24 DNA Artificial Sequence synthetic oligonucleotide 11gatccatcga tctcgagatc gatc 24 12 28 DNA Artificial Sequence syntheticoligonucleotide 12 tgatttccaa gcgcgctgag atcaccag 28 13 28 DNAArtificial Sequence synthetic oligonucleotide 13 tgagcaacga agcggctgagatcaccag 28 14 5 PRT Rhizomucor miehei 14 Tyr Tyr Phe Trp Asp 1 5 15 17DNA Rhizomucor miehei 15 gcrtcccara artarta 17 16 18 PRT Humicola griseaMISC_FEATURE (15)..(15) the x at position 15 can be any amino acid. 16Ala Ala Val Asp Thr Phe Ile Asn Thr Glu Lys Pro Ser Ala Xaa Asn 1 5 1015 Ser Leu 17 26 PRT Humicola grisea MISC_FEATURE (14)..(14) the x atposition 14 can be any amino acid. 17 Pro Leu Trp Ser Ile Thr Val ProIle Lys Ala Thr Gly Xaa Ala Val 1 5 10 15 Gln Tyr Lys Tyr Ile Lys ValXaa Gln Leu 20 25 18 25 PRT Humicola grisea MISC_FEATURE (17)..(17) thex at position 17 can be any amino acid. 18 Ala Ala Val Arg Pro Leu IleAsn Pro Glu Lys Pro Ile Ala Trp Asn 1 5 10 15 Xaa Leu Lys Ala Asn IleGly Pro Asn 20 25 19 39 PRT Humicola grisea 19 Ile Asn Thr Glu Lys ProIle Ala Trp Asn Lys Leu Leu Ala Asn Ile 1 5 10 15 Gly Pro Asn Gly LysAla Ala Pro Gly Ala Ala Ala Gly Val Val Ile 20 25 30 Ala Ser Pro Ser ArgThr Asp 35 20 17 DNA Artificial Sequence synthetic oligonucleotideprobes 20 cartayaart ayathaa 17 21 6 PRT Artificial Sequence syntheticoligonucleotide probes 21 Gln Tyr Lys Tyr Ile Lys 1 5 22 17 DNAArtificial Sequence synthetic oligonucleotide probes 22 cartayaartatattaa 17 23 17 DNA Artificial Sequence synthetic oligonucleotideprobes 23 cartayaart acattaa 17 24 17 DNA Artificial Sequence syntheticolibonucleotide probe 24 cartayaart atatcaa 17 25 17 DNA ArtificialSequence synthetic oligonucleotide probe 25 cartayaart acatcaa 17 26 17DNA Artificial Sequence synthetic oligonucleotide probe 26 cartayaartatataaa 17 27 17 DNA Artificial Sequence synthetic oligonucleotide probe27 cartayaart acataaa 17 28 17 DNA Artificial Sequence oligonucleotideprobe 28 cagtacaagt atatcaa 17

What is claimed is:
 1. A vector comprising a DNA sequence capable ofexpressing a heterologous polypeptide in a filamentous fungus and ofcausing secretion of said heterologous polypeptide from said filamentousfungus.
 2. The vector of claim 1 wherein said secreted heterologouspolypeptide is biochemically active.
 3. The vector of claim 1 whereinsaid heterologous polypeptide is an enzyme.
 4. The vector of claim 3wherein said enzyme is selected from the group consisting of chymosin,prochymosin, preprochymosin, A. niger glucoamylase, Humicola griseaglucoamylase and Mucor mehei carboxyl protease.
 5. A DNA sequenceencoding a heterologous polypeptide, said DNA sequence being capable ofbeing expressed in a filamentous fungus and of causing secretion of saidheterologous polypeptide.
 6. The DNA sequence of claim 5 wherein saidsecreted heterologous polypeptide is biochemically active.
 7. The DNAsequence of claim 5 wherein said heterologous polypeptide is an enzyme.8. The DNA sequence of claim 7 wherein said enzyme is selected from thegroup consisting of chymosin, prochymosin, preprochymosin, A. nigerglucoamylase, Humicola grisea glucoamylase and Mucor mehei carboxylprotease.
 9. A vector for transforming a filamentous fungus comprising aDNA sequence encoding a heterologous polypeptide and a DNA sequenceencoding a signal sequence operably linked thereto, said signal sequencebeing functional in a secretory system in said filamentous fungus. 10.The vector of claim 9 wherein said signal sequence is native to saidheterologous polypeptide.
 11. The vector of claim 10 wherein said nativesignal sequence and said heterologous sequence are selected from thegroup consisting of DNA sequences encoding bovine preprochymosin, Mucormeihei preprocarboxyl protease and A. niger preproglucoamylase.
 12. Thevector of claim 9 wherein said signal sequence is foreign to saidheterologous polypeptide.
 13. The vector of claim 12 wherein saidforeign signal sequence is selected from the group consisting of DNAsequences encoding the signal sequence of bovine preprochymosin, A.niger glucoamylase and Mucor miehei carboxyl protease.
 14. The vector ofclaim 9 further comprising a DNA sequence encoding a promoter sequencefunctionally recognized by said filamentous fungus includingtranscription and translation control sequences operably linked to saidDNA sequence encoding said signal sequence.
 15. The vector of claim 14wherein said DNA sequence encoding said promoter sequence is selectedfrom the group consisting of DNA sequences encoding the promotersequence of A. niger glucoamylase and Mucor miehei carboxyl protease.16. The vector of claim 9 further comprising DNA sequences encoding afunctional polyadenylation sequence operably linked to said DNA sequenceencoding said heterologous polypeptide.
 17. The vector of claim 16wherein said polyadenylation sequence is selected from the groupconsisting of DNA sequences encoding the polyadenylation sequence of A.niger glucoamylase or Mucor meihei carboxyl protease.
 18. The vector ofclaim 9 further comprising a DNA sequence encoding a selectioncharacteristic expressible in said filamentous fungus.
 19. The vector ofclaim 18 wherein said selection characteristic is selected from thegroup consisting of DNA sequences encoding N. crassa pyr4, A. nidulansacetamidase, A. nidulans argB and A. nidulans trpC.
 20. The vector ofclaim 9 further comprising a DNA sequence capable of increasing thetransformation efficiency of said vector into said filamentous fungus.21. The vector of claim 20 wherein said DNA sequence for increasingfungal transformation efficiency is ANS-1.
 22. The vector of claim 9wherein said secreted heterologous polypeptide is an enzyme.
 23. Thevector of claim 22 wherein said enzyme is selected from the groupconsisting of chymosin, prochymosin, preprochymosin, A. nigerglucoamylase, Humicola grisea glucoamylase and Mucor mehei carboxylprotease.
 24. The vector of claim 9 wherein said secreted heterologouspolypeptide is a mammallian polypeptide.
 25. The vector of claim 9wherein said secreted heterologous polypeptide is biochemically active.26. A filamentous fungus containing a vector selected from the groupconsisting of the vectors of claims 1 through
 25. 27. A filamentousfungus capable of expressing and secreting a heterologous polypeptide.28. The filamentous fungus of claim 27 wherein said heterologouspolypeptide is integrated into the geneome of said filamentous fungus.29. The filamentous fungus of claim 27 wherein said filamentous fungusis selected from the group consisting of Aspergillus species,Trichoderma species and Mucor species.
 30. The filamentous fungus ofclaim 27 wherein said filamentous fungus is A. nidulans or A. awamori.31. The filamentous fungus of claim 27 wherein said filamentous fungusis Trichoderma reesei.
 32. The filamentous fungus of claim 27 whereinsaid heterologous polypeptide is an enzyme selected from the groupconsisting of chymosin, prochymosin, preprochymosin, A. nigerglucoamylase, Humicola grisea glucoamylase and Mucor mehei carboxylprotease.
 33. The filamentous fungus of claim 27 wherein said secretedheterologous polypeptide is a mammalian polypeptide.
 34. The filamentousfungus of claim 27 wherein said secreted heterologous polypeptide isbiochemically active.
 35. A process for making a heterologouspolypeptide comprising: transforming a filamentous fungus with a vectorcontaining DNA sequences capable of expressing a heterologouspolypeptide and of causing secretion of said heterologous polypeptidefrom said filamentous fungus, and expressing and secreting saidheterologous polypeptide.
 36. The process of claim 35 wherein saidexpressing and secreting is carried out in a culture medium comprisingutilizable carbon, nitrogen and phosphate sources, surfactant and traceelements.
 37. The process of claim 35 further comprising the step ofisolating said secreted heterologous polypeptide.
 38. The process ofclaim 35 wherein said vector containing DNA sequences capable ofexpressing a heterologous polypeptide and of causing secretion of saidheterologous polypeptide is selected from the group consisting of thevectors of claims 1 through
 25. 39. The process of claim 35 wherein saidsecreted heterologous polypeptide is an enzyme selected from the groupconsisting of chymosin, prochymosin, preprochymosin, A. nigerglucoamylase, Humicola grisea glucoamylase and Mucor mehei carboxylprotease.
 40. The process of claim 35 wherein said secreted heterologouspolypeptide is a mammalian polypeptide.
 41. The process of claim 35wherein said secreted heterologous polypeptide is biochemically active.